Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase

ABSTRACT

A nucleic acid fragment encoding a herbicide-resistant plant acetolactate synthase protein is disclosed. This nucleic acid fragment contains at least one nucleotide mutation resulting in one amino acid change in one of seven substantially conserved regions of acetolactate synthase amino acid homology. This mutation results in the production of an acetolactate synthase protein which is resistant to sulfonylurea herbicide compounds compared to the wild-type protein. Transformation of herbicide sensitive plants or plant cells with the fragment results in resistance to the herbicide.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/892,305filed Jun. 2, 1992, now U.S. Pat No. 5,378,824, which is a divisional ofapplication U.S. Ser. No. 07/642,976 filed Jan. 18, 1991 (and now issuedas U.S. Pat. No. 5,141,870), which is a divisional of application U.S.Ser. No. 07/164,360 filed Mar. 4, 1988 (and now issued as U.S. Pat. No.5,013,659), which is a continuation-in-part of application U.S. Ser. No.06/900,609 filed Aug. 26, 1986, now abandoned.

TECHNICAL FIELD

The present invention relates to nucleic acid fragments encoding aherbicide-resistant form of the enzyme acetolactate synthase (ALS).

BACKGROUND

Sulfonylurea herbicides such as sulfometuron methyl (I) andchlorsulfuron (II) inhibit growth of some bacteria, yeast and higherplants by blocking acetolactate synthase [ALS, EC 4.1.3.18], the firstcommon enzyme in the biosynthesis of the branched-chain amino acidsvaline, leucine and isoleucine. The biosynthesis of branched-chain aminoacids and, hence, the toxicity of sulfonylurea herbicides is restrictedto plants and microbes. ALS is also inhibited by a structurallyunrelated class of herbicides, the imidazolinones. ##STR1##

Three major isozymes of ALS, designated I, II and III, have beenidentified in enteric bacteria. Isozymes I and III, but not II, aresensitive to end-product inhibition by valine. Each of the threebacterial isozymes comprises a large and a small protein subunit. ALSenzymes from the yeast Saccharomyces cerevisiae and from some higherplants have been partially characterized and show some degree ofend-product inhibition. It is not known if the yeast and plant ALSenzymes consist of one or more different polypeptides. Evidence suggeststhat the cellular locations of the yeast and plant ALS enzymes are inthe mitochondria and chloroplasts, respectively.

Genes encoding ALS enzymes have been isolated from the enteric bacteriaSalmonella typhimurium and Escherichia coli, and the yeast S.cerevisiae. The nucleotide sequences of the genes coding for the twosubunits of E. coli, ALS isozymes I, II and III show that they areorganized as operons ilvBN, ilvGM and ilvIH, respectively. Comparison ofthe deduced amino acid sequences of the large subunits of the E. coliALS isozymes shows three regions with about 50% conserved amino acids,comprising about two-thirds of the proteins, and separated by regionssharing little discernible homology. Amino acid sequence conservation,though less extensive, is also evident among the small subunits of thebacterial isozymes. In the yeast S. cerevisiae, a single gene, ILV2,essential for ALS activity was identified. Nucleotide sequence analysisof the ILV2 gene has revealed that the polypeptide encoded by it ishomologous to the large subunits of the bacterial ALS isozymes. Thededuced amino acid sequence of the yeast ALS shows the same degree ofstructural organization and the same degree of homology as is observedbetween the large subunits of the bacterial isozymes, except for aboutninety amino acids at the amino terminus of the yeast protein that arebelieved to be involved in the translocation of the protein into themitochondrion. No information on the structure of plant genes encodingALS or the amino acid sequence of plant ALS enzymes was available priorto the inventions disclosed herein.

Enteric bacterial isozyme I is the only ALS in nature that is known tobe insensitive to inhibition by sulfometuron methyl and chlorsulfuron.Therefore, enteric bacteria are sensitive to these herbicides only inthe presence of valine, which inhibits isozyme I. Sulfonylureaherbicide-resistant mutant forms of the enteric bacteria Salmonellatyphimurium and E. coli (selected in the presence of valine), the yeastS. cerevisiae and the higher plants Nicotaiana tabacum (tobacco),Arabidopsis thaliana and Zea mays (corn) have been identified. Thesemutant phenotypes cosegregate with herbicide-resistant forms of ALSthrough genetic crosses. In S. typhimurium the herbicide-resistancemutations are genetically linked to a gene encoding ALS, and in E. coliand S. cerevisiae, these mutations reside in the structural genes forALS. In the higher plants the mutations responsible for the resistanceare inherited as single, dominant or semidominant nuclear traits. Intobacco, these mutations map to either of two unlinked genetic loci.

The chemical control of undesirable weeds associated with agronomicallyuseful crops requires the use of highly selective chemical herbicides.In some cases, it is difficult to identify any chemical which killsweeds without injury to the crop plant. The introduction ofherbicide-resistance as a biological trait in crop plants would overcomethis difficulty.

Although many genes involved in the structure and function ofdifferentiated plant tissues and organs are not expressed inundifferentiated tissues, those involved in basic cellular functions areexpressed and can be selected for in a disorganized callus or cellsuspension culture. This has been demonstrated in many cases by theselection of a phenotype in tissue culture from which plants expressingthe same phenotype have been regenerated. Examples include the in vitroselection of plants resistant to herbicides, pathotoxins or diseases,antibiotics, amino acid analogues, salt tolerance, etc.

Since acetolactate synthase is an enzyme involved in the basic cellularmetabolic activity of amino acid biosynthesis, it was expected and hasbeen demonstrated that genes encoding this enzyme are expressed incallus tissue as well as the whole plant. The sulfonylurea resistanttobacco mutants described in this patent, S4, C3 and Hra, were firstselected in tissue culture and subsequently regenerated into wholeplants in which the resistant phenotypes were retained in a geneticallystable manner. Callus tissues derived from regenerated plants or theirprogeny continue to grow on concentrations of the herbicide whichinhibit the growth of wild type callus. Thus resistance to asulfonylurea herbicide at the plant cellular level is predictive ofresistance at the whole plant level. In addition, it has beendemonstrated in bacteria, yeast and higher plants that mutationsresulting in the production of herbicide resistant ALS are sufficient toconfer resistance at the cellular level and, in the case of plants, atthe whole plant level. Therefore, the observation of herbicide-resistantALS in extracts of plant cells is also predictive of herbicide resistantgrowth of cultured plant cells and herbicide resistant growth of wholeplants.

Sulfonylurea herbicide resistant mutant tobacco and corn plants havebeen obtained by regeneration from mutant tissue culture cell lines andresistant Arabidopsis plants have been produced by seed mutagenesis.There are, however, significant advantages to be derived from isolationof a nucleic acid fragment able to confer herbicide resistance and itssubsequent introduction into crops through genetic transformation. Onecan obtain cross species transfer of herbicide resistance, whileavoiding potential limitations of tissue culture, seed mutagenesis, andplant breeding as techniques to transfer novel DNA fragments and traits.Plants exhibiting herbicide resistance achieved through transformationwith a mutant ALS gene may possess distinct advantages relative to thoseregenerated after selection with a herbicide in tissue culture. Theinsertion of an additional gene or genes encoding an altered form of theALS enzyme in the transformed plant can supply additional plantmetabolic capabilities. It can also enable the plant molecular biologistto engineer desired selectivities into the added gene(s). Further, theinsertion of the additional gene(s) in particular locations can resultin enhanced levels of expression of the mutant ALS enzyme, as well as indifferent patterns of tissue or temporal expression of the gene. Suchchanges may result in production of new protein in root systems, forexample. Tissue specific and/or temporal expression of the introducedgene can also be modulated through the substitution of specific generegulatory sequences for the native gene regulatory sequences. Suchsubstitutions can, for example, place gene expression under the controlof chemical inducing agents. Finally, control of the chromosomallocation of the inserted gene may avoid the complications of the nativegene being linked to a disadvantageous allele which would requireextensive plant breeding efforts to subsequently separate the traits.And, the absence of exposure of the plant tissues to mutagenic agentsobviates the need for extensive back-crossing to remove undesirablemutations generated by these agents.

Although genes isolated from one plant have been introduced andexpressed in other plants, non-plant genes have been expressed in plantsonly as chimeric genes in which the coding sequences of the non-plantgenes have been fused to plant regulatory sequences required for geneexpression. However, it would be difficult to introduce herbicideresistance into plants by introducing chimeric genes consisting ofbacterial or yeast genes for herbicide-resistant forms of ALS, since (a)these microbial ALS enzymes are believed to lack a specific signal(transit) peptide sequence required for uptake into plant chloroplasts,the cellular location of plant ALS, (b) the bacterial isozymes consistof two different polypeptide subunits, and (c) the microbial ALS enzymesmay not function optimally in the foreign cellular environment of higherplants. Therefore, there is a need for nucleic acid fragments (1) whichencode a herbicide-resistant form of plant ALS, and (2) which can conferherbicide resistance when introduced into herbicide sensitive plants.

SUMMARY OF THE INVENTION

The present invention provides a nucleic acid fragment comprising anucleotide sequence encoding plant acetolactate synthase. The nucleotidesequence comprises at least one sequence which encodes one of thesubstantially conserved amino acid subsequences designated A, B, C, D,E, F and G in FIG. 6. The nucleic acid fragment is further characterizedin that at least one of the following conditions is met,

a) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence A wherein ε₁ is an amino acid other than alanine, or ε₂ isan amino acid other than glycine,

b) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence B wherein α₁ is an amino acid other than proline,

c) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence C wherein δ₂ is an amino acid other than alanine,

d) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence D wherein λ₁ is an amino acid other than lysine,

e) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence E wherein γ₁ is an amino acid other than aspartic acid,

f) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence F wherein β₃ is an amino acid other than tryptophan, or β₈is an amino acid other than valine or β₇ is an amino acid other thanphenylalanine, and

g) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence G wherein σ₁ is an amino acid other than methionine.

In another embodiment, the instant invention provides a nucleic acidfragment encoding plant acetolactate synthase which is capable of beingincorporated into a nucleic acid construct used to transform a plantcontaining wild-type acetolactate synthase which is sensitive to asulfonylurea herbicide compound, said nucleic acid fragment having atleast one point mutation relative to the wild-type nucleic acid fragmentencoding plant acetolactate synthase such that upon transformation withsaid nucleic acid construct said plant contains said nucleic acidfragment and renders said plant resistant to the application of saidsulfonylurea herbicide compound.

In another embodiment, the present invention provides an acetolactatesynthase protein which is resistant to a sulfonylurea herbicide compoundcomprising an amino acid sequence wherein a substitution of at least oneamino acid has occurred.

In still another embodiment, the present invention provides nucleic acidconstructs, monocotyledonous and dicotyledonous plants, and tissuecultures which contain the specified nucleic acid fragment. Theinvention further provides methods for transforming plants with thespecified fragments, selecting transformed plant cells, and growingtransformed plants.

The present invention also provides a method for selecting plant cellstransformed with the nucleic acid fragment of the present invention Themethod comprises introducing the fragment into plant cells whose growthis sensitive to inhibition by herbicides to which the ALS encoded by thefragment is resistant to form a transformed plant cell. The transformedplant cells whose growth is resistant to the selected herbicide areidentified by selection at a herbicide concentration which inhibits thegrowth of the untransformed plant cells.

In another aspect, the present invention is a method for controllingunwanted vegetation growing at a locus where a herbicide-resistant,agronomically useful plant (transformed with the nucleic acid fragmentof the present invention) has been cultivated. The method comprisesapplying to the locus to be protected an effective amount of herbicide.In still another aspect, the present invention provides a nucleic acidfragment comprising the linkage of a nucleic acid fragment encodingacetolactate synthase conferring herbicide resistance and a secondnucleic acid fragment conferring a second trait wherein said nucleicacid fragment is utilized to transform a plant and the expression ofherbicide resistance by said plant upon application of sulfonylureacompound is utilized to detect the presence of said second nucleic acidfragment in said plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a physical map of nucleic acid insert fragments containing ALSgenes isolated from a genomic library of DNA from the tobacco Hramutant.

FIG. 2 is a diagram of plasmid pAGS152 showing a physical map of thenucleic acid fragment from tobacco encoding a herbicide-resistant ALS.

FIG. 3 is a physical map of a nucleic acid insert fragment in phageclone 35 isolated from genomic library of DNA from the tobacco C3mutant.

FIGS. 4A-4D are a nucleotide sequence, and the cognate deduced aminoacid sequence, of a gene from the Hra mutant of tobacco encoding aherbicide-resistant form of ALS from tobacco.

FIGS. 5A-5D are a nucleotide sequence, and the cognate deduced aminoacid sequence, of a gene from the C3 mutant of tobacco encoding aherbicide-resistant form of ALS.

FIGS. 6A-6F are a comparison of deduced amino acid sequences of thelarge subunits of bacterial ALS and the yeast and plant ALS enzymes.

FIG. 7 is a physical map of a nucleic acid insert fragment andsub-fragments derived from phage clone 21 isolated from a genomiclibrary of sugarbeet DNA.

FIGS. 8A-8C are a comparison of deduced amino acid sequences of plantALS enzymes.

FIG. 9 is a diagram of plasmid pKAR showing a physical map of thenucleic acid fragment from Arabidopsis encoding a herbicide-resistantALS.

FIGS. 10A-10D are a nucleotide sequence, and the cognate deduced aminoacid sequence, of a gene from Arabidopsis encoding a herbicide-resistantform of ALS.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides specified nucleic acid fragments whichconfer herbicide resistance when introduced into herbicide-sensitiveplants. As used herein, the term "nucleic acid fragment" refers to alinear segment of single- or double-stranded deoxyribonucleic acid (DNA)or ribonucleic acid (RNA), which can be derived from any source.Preferably, the nucleic acid fragment of the present invention is asegment of DNA. The term "plant" refers to a photosynthetic organismincluding algae, mosses, ferns, gymnosperms, and angiosperms. The term,however, excludes prokaryotic and eukaryotic microorganisms such asbacteria, yeast, and fungi. "Plant cell" includes any cell derived froma plant, including undifferentiated tissue such as callus or gall tumor,as well as protoplasts, and embryonic and gametic cells. The term "plantacetolactate synthase" refers to the specified enzyme when expressed ina plant or a plant cell. The term "nucleotide sequence" refers to apolymer of DNA or RNA which can be single- or double-stranded,optionally containing synthetic, non-natural,or altered nucleotidescapable of incorporation into DNA or RNA polymers. As used herein, theexpression "substantially conserved amino acid sequences" refers toregions of amino acid homology between polypeptides comprising ALSenzymes from different sources. In the present invention sevensubstantially conserved amino acid sequences, designated A, B, C, D, E,F, and G are shown in FIG. 6. One skilled in the art could align theamino acid sequences of ALS enzymes from different sources to theschematic of FIG. 6 to identify the segments therein which are thesubstantially conserved amino acid sequences defined herein. The skilledperson could then determine whether the identified segments have thecharacteristics disclosed and claimed in the present application. It isto be understood that the expression includes modifications of thesegments which do not adversely affect the activity of the ALS enzyme.The term "nucleic acid construct" refers to a plasmid, virus,autonomously replicating sequence, phage or linear segment of a single-or double-stranded DNA or RNA, derived from any source, which is capableof introducing a nucleic acid fragment into a biological cell.

"Regulatory nucleotide sequence", as used herein, refers to a nucleotidesequence located 5' and/or 3' to a nucleotide sequence whosetranscription and expression is controlled by the regulatory nucleotidesequence in conjunction with the protein synthetic apparatus of thecell. As used herein, a "regulatory nucleotide sequence" can include apromoter region, as that term is conventionally employed by thoseskilled in the art. A promoter region can include an association regionrecognized by an RNA polymerase, one or more regions which control theeffectiveness of transcription initiation in response to physiologicalconditions, and a transcription initiation sequence.

"Transit peptide" refers to a signal polypeptide which is translated inconjunction with a polypeptide encoded by a product nucleotide sequence,forming a polypeptide precursor. In the process of transport to aselected site within the cell, for example, a chloroplast, the transitpeptide can be cleaved from the remainder of the polypeptide precursorto provide an active or mature protein.

"Herbicide," as used herein, refers to an antibiotic compound whichinhibits the metabolism, growth, or replication of plant cells or wholeplants. Cells transformed with a construct of the present inventionexhibit selectable cross-resistance to certain structurally relatedsulfonamide compounds effective as broad-spectrum preemergent andpost-emergent herbicides. As used herein in a generic sense,"sulfonylurea herbicides" refer toN-(heterocyclicaminocarbonyl)arylsulfonamide compounds exhibitingbroad-spectrum herbicidal activity and low mammalian toxicity."Selective concentration" refers to a concentration of an inhibitor orantibiotic compound, for example, a herbicide, which is capable ofinhibiting the metabolism, growth, or multiplication of a wild-type cellor organism. Such an organism, as well as clones thereof, is referred toas a "sensitive" organism or cell. "Resistance" refers to a capabilityof an organism or cell to grow in the presence of selectiveconcentrations of an inhibitor. In relation to particular enzymes orproteins, "sensitive" indicates that the enzyme or protein issusceptible to specific inhibition by a particular inhibiting compound,for example, an antibiotic or herbicide. In relation to particularenzymes or proteins, "resistant" indicates that the enzyme or protein,as a result of a different chemical structure, expresses activity in thepresence of a selective concentration of a specific inhibitor whichinactivates sensitive variants of the enzyme or protein. The term"selectable genetic marker" refers to a nucleotide sequence which, whenincorporated into the genome of an organism, allows growth of thatorganism and its progeny under conditions which inhibit growth of theorganism lacking the selectable genetic marker. For example, a genewhich encodes an enzyme that is resistant to specific inhibition by aparticular antibiotic compound, such as a herbicide, can function as aselectable genetic marker by allowing an organism, such as a plant, togrow and propagate in the presence of a selective concentration of thecompound. A second nucleic acid fragment, controlling a property whichis difficult to assay, can be covalently linked to the selectablegenetic marker, in which case the presence of the selectable marker,indicated by growth of an organism under selective conditions, can beused to detect an organism containing the second nucleic acid fragment.

Preparation of DNA Fragments Encoding Herbicide-Resistant ALS

Callus cultures of sensitive tobacco (Nicotiana tabacum vat. Xanthi)were exposed to sulfometuron methyl at 2 ppb according to the methoddescribed by Chaleff, U.S. Pat. No. 4,443,971. Resistant cell linesdesignated C3 and S4 were selected. Standard genetic analysis of plantsregenerated from these cell lines indicated that the C3 and S4 lineseach carried a single semi-dominant nuclear gene mutation responsiblefor the herbicide resistance trait and that the C3 and S4 mutations werenot genetically linked, i.e. were in different genes designated SURA andSURB, respectively. The C3 and S4 lines were shown to produce ALS enzymeactivity one-hundred fold more resistant to the sulfonylurea herbicideschlorsulfuron and sulfomenturon methyl than ALS from wild type.Production of herbicide resistant ALS activity cosegregated in geneticcrosses with resistance to growth inhibition by the herbicides. Theobservation of two different genes that had mutated to form herbicideresistant ALS was not unexpected because N. tabacum is believed to be anallotetraploid plant formed from N. tomentosiformis and N. sylvestris,essentially containing two complete genomes. Thus, the S4 and C3 celllines each contain one mutant and one wild type ALS gene. The S4 cellline was exposed to sulfometuron methyl at 200 ppb, a selectiveconcentration which completely inhibits the growth of S4. Cell linesresistant to 200 ppb were identified; one such line was designated Hra.Hra was shown to tolerate concentrations of sulfometuron methyl onethousand times greater than that required to completely inhibit thegrowth of wild type callus. Hra was shown to be cross resistant tochlorsulfuron. Plants were regenerated from Hra callus cultures. Geneticanalysis of the plants demonstrated that the Hra and S4 mutations werelinked indicating that the Hra line contained a second mutation in themutant gene of the progenitor S4 line. ALS activity in extracts ofleaves of wild type and homozygous Hra mutant tobacco plants wasdetermined. The ALS activity in the extract from Hra mutant plants wasabout one thousand fold more resistant to chlorsulfuron than was theactivity of the wild type plants. Hra mutant plants were further shownto be cross resistant to the foliar application of the followingcompounds:

2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylicacid, (1-methylethanamine) salt;

5-ethyl-4,5-dihydro-2-[4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylicacid;

2-(2-chloroethoxy)-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide;

2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide;

2-[[(4-chloro-6-methoxypyrimidin-2-yl)aminocarbonyl]aminosulfonyl]benzoicacid, ethyl ester;

N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-2,3-dihydro-2-methylbenzo[.beta.]thiophene-7-sulfonamide,1,1-dioxide;

7-chloro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-3,4-dihydro-2-methyl-2H-1,2-benzothiazine-8-sulfonamide,S,S-dioxide;

2-[[(4-methoxy-6-methylpyrimidin-2-yl)aminocarbonyl]aminosulfonyl]-6-methylbenzoicacid, methyl ester;

5,7-dimethyl-N-(2-methyl-6-nitrophenyl)[1,2,4]-triazolo[1,5-A]pyrimidin-2-sulfonamide;

2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-quinolinecarboxylicacid;

6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluic acid andp-toluic acid, methyl esters;

2-[[(4,6-dimethylpyrimidin-2-yl)aminocarbonyl]aminosulfonyl]benzoicacid, methyl ester;

N-(2,6-dichlorphenyl)-5,7-dimethyl[1,2,4]triazolo[1,5-A]pyrimidin-2-sulfonamide;

N-(2-chloro-6-methylphenyl)-5,7-methyl[1,2,4]triazolo[1,5-A]pyrimidin-2-sulfonamide.

In order to clone a herbicide resistant ALS gene, tobacco DNA wasisolated from the S4 homozygous mutant line of Nicotiana tabacum. 50 gportions of callus tissue were frozen in liquid N₂, and thenlyophilized. The resulting dried tissue was then ground at about 23° C.in a blender, using 15 second bursts, until powdered. Ten volumes of asucrose buffer (0.3 M sucrose, 50 mM Tris-HCl pH 8.0, 5 mM MgCl₂) wereadded, and the resulting suspension was incubated at 0° C. for 5minutes. The suspension was then filtered through cheesecloth, andcentrifuged at 350×g for 10 minutes. The nuclear pellet was thenresuspended in lysis buffer (20 mM EDTA, 50 mM Tris-HCl pH 8.0, 1%Sarkosyl), CsCl added to provide 0.95 g per mL buffer, and the resultingmixture centrifuged at 17,000×g for 20 minutes at 4°. Ethidium bromidewas added to the resulting supernatant to a concentration of 400 μg permL, the refractive index was adjusted to 1.39, and the resultingsolution centrifuged at 90,000×g in a Beckman Ti70 rotor at 20° C. for 3days. The resulting fluorescent DNA band was removed from the gradient,and treated with isopropanol to extract the ethidium bromide. Finally,the DNA was dialyzed against TE buffer and precipitated by addition ofethanol.

A Nicotiana genomic library was prepared from this DNA as follows, usingthe phage lambda vector EMBL4 described by Frischauf et al., J. Mol.Bio. 170:827 (1983). EMBL4 phage was prepared from agarose plate stocksprepared by the method of Davis et al; Advanced Bacterial Genetics,(Cold Spring Harbor Laboratory, New York, 1980). Phage DNA was preparedas described by Silhavy et al., Experiments with Gene Fusions, (ColdSpring Harbor Laboratory, New York, 1984), by concentrating phage withpolyethylene glycol, removing the polyethylene glycol by chloroformextraction, and purifying phage using glycerol step gradients. Theresulting purified phage was then treated with deoxyribonuclease andribonuclease prior to phenol extraction. Phage DNA was spooled fromethanol. To prepare arms of the EMBL4 phage, phage DNA was sequentiallydigested with Sal I and Bam HI endonucleases. The arms were annealed andthen separated from the central fragment on a 10-40% sucrose gradient,as described by Maniatis et al., Molecular Cloning: A Laboratory Manual.(Cold Spring Harbor Laboratory, New York, 1982). The arms werecompletely denatured and reannealed prior to ligation to tobacco DNA.Tobacco DNA, prepared as previously described, was partially digestedwith Sau3A endonuclease and sedimented through a 10-40% sucrosegradient. Fractions from the sucrose gradient were then analyzed byelectrophoresis on 0.5% agarose gels. Fractions containing fragments inthe 20-40 kb size range were dialyzed, precipitated, and ligated to thelambda phage DNA arms. The DNA was ligated at a concentration of 135 μgper mL vector and 45 μg per mL insert DNA. The resulting ligatedconcatamers were then packaged using lambda DNA packaging extracts. Theresulting yield of phage was approximately 4.5×10⁵ phage per μg insertDNA. A library of approximately 400,000 phage was constructed,representing an estimated 99% complete library for tobacco, which has anapproximate genomic content of 1.65 picograms, or 1.52×10⁹ base pairs(Zimmerman, et al., Chromosoma 59:227 1977).

The resulting phage library of Nicotiana DNA was grown and plated on E.coli stain LE392 (ATCC 33572), as disclosed by Silhavy et al.,Experiments with Gene Fusions, (Cold Spring Harbor Laboratory, New York,1984). Phage were plated to provide 2000-5000 plaques on 90 mm petridishes or 50,000 plaques on 150 mm petri dishes. Plaque lifts were doneby the method of Benton et al., Science 196:180 (1977). Followingtransfer of phage DNA to nitrocellulose filters, the filters wereprehybridized by incubation for about 4 hours at 56° C. in 6×SSPEcontaining 0.5% SDS, 100 μg per mL denatured calf thymus DNA, and10×Denhardt's solution. Hybridization was then accomplished as describedby Maniatis, et al., Molecular Cloning: A Laboratory Manual, (ColdSpring Harbor Laboratory, New York, 1982) p. 326. In this step, a freshaliquot of hybridization solution was added, together with about 10⁸ cpmof the radioactive yeast ALS gene probe. Hybridization was allowed tooccur for about 24-48 hours at 56° C. At this point, the filters werefirst rinsed for about 4 hours in 6×SSPE at 56° C., then rinsed threeadditional times for 20 minutes each in 2×SSPE at about 23° C. Thefilters were than dried and exposed at -70° C. for 2 days, using KodakXAR or XRP x-ray film and a Du Pont Cronex® Lightning Plus™ intensifyingscreen. Exposed spots on the film indicated the position of plaquespotentially containing Nicotiana ALS genes.

The autoradiograms prepared as described above were then oriented overthe original bacteriophage-containing petri dishes. Using the wide endof a sterile Pasteur pipette, plaques corresponding to the darkest spotson the autoradiograms were excised. The plaques selected were theneluted into SM buffer and plated onto fresh 90 mm petri dishes. Eachdish received about 100-200 phage. The complete phage location processwas then reiterated, using freshly prepared probe. In this manner, thephage location and isolation steps were repeated until the majority ofplaques indicated the presence of phage containing DNA capable ofhybridization to the yeast ALS gene probe.

Mini-preparations of DNA from the plaque-purified phage designated NtA13were isolated as described and worked up as described by Maniatis et al,Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,New York, 1982), p. 371. EcoRI restriction endonuclease digests of theDNA mini-preparations were electrophoresed through 0.7% agarose gels andblotted onto nitrocellulose filters. Fragments containing the ALS genewere then identified by hybridization with the yeast ALS gene probe.Fragments capable of hybridization to the probe were then isolated andsubcloned into vectors pBR322, M13mp9, or M13mp18. These fragments werethen sequenced using oligonucleotide primers in a dideoxy chaintermination procedure conducted substantially as described by Sanger etal., Proc. Natl. Acad. Sci. USA 74:5463 (1977). A kit available from NewEngland Biolabs (Beverly, Mass., U.S.A.) was employed. Use of syntheticoligonucleotide primers allowed extension of a DNA sequence along acloned fragment in overlapping segments. Computer analysis of the DNAsequence identified a 667 codon open reading frame. The deduced aminoacid sequence of this open reading frame was substantially homologous tothe sequences previously determined from the Sacharomyces cerevisiaeILV2 gene and the E. coli ilvG gene, indicating that the DNA fragmentrecovered from the Nicotiana genomic library contained a tobacco ALSgene. To determine whether this ALS gene encoded the wild type herbicidesensitive enzyme or the mutant herbicide resistant enzyme from the S4line, the gene was introduced into wild type herbicide sensitive tobaccoby Agrobacterium tumefaciens mediated transformation.

The results shown in Table 1 indicated that transformation of tobaccohad been achieved based on production of kanamycin resistant callus. Thekanamycin resistant callus remained sensitive to the sulfonylureaherbicide chlorsulfuron, whether or not the tobacco ALS gene waspresent, indicating that the ALS gene isolated from the tobacco S4mutant in phage Nta 13 encoded the wild type herbicide sensitive enzyme.This plant ALS gene has been used as a DNA hybridization probe toisolate other plant ALS genes, including genes which encode herbicideresistant ALS, and has been mutagenized in vitro to encode herbicideresistant forms of ALS.

                  TABLE 1                                                         ______________________________________                                        Results from Callus Tests of                                                  GVKNT13 Infected Tobacco                                                      Number of transformed shoot explants producing callus on                      selective and non-selective media.                                                       GVKNT13.sup.1                                                                           GVKK.sup.2                                                                              GV3850.sup.3                                   ______________________________________                                        Exp. #1                                                                       No selection 59/62       12/13     10/10                                      Kanamycin, 50 mg/L                                                                         53/62        8/13     0/10                                       Chlorsulfuron, 10 ppb                                                                      0/62         0/13     0/10                                       Exp. #2                                                                       No selection 96/102      21/23     22/25                                      Kanamycin, 50 mg/L                                                                         81/102      16/23     0/25                                       Chlorsulfuron, 10 ppb                                                                       0/102       0/23     0/25                                       ______________________________________                                         .sup.1 Agrobacterium strain containing Ti plasmid carrying tobacco ALS        gene and NOS/NPTII gene (Kanamycin resistance)                                .sup.2 Agrobacterium strain containing Ti plasmid carrying only NOS/NPTII     gene                                                                          .sup.3 Agrobacterium strain containing Ti plasmid (devoid of either           tobacco ALS or NOS/NPTII genes)                                          

A genomic library of DNA from the Hra mutant of tobacco was made inbacteriophage lambda and screened for clones that hybridized to the wildtype tobacco ALS gene from the S4 mutant. Several phage clones wereisolated. Physical mapping of the tobacco DNA inserts using restrictionendonucleases revealed the presence of two distinct classes of DNAfragments representative of the two tobacco ALS genes SURA and SURB.Comparison of the physical maps of the SURA and SURB genes of N. tabacumto maps from the progenitor species showed that the SURA gene came fromN. sylvestris and the SURB gene came from N. tomentosiformis. The wildtype ALS gene isolated previously from the S4 mutant was designatedSURA. The genetic linkage of the high level herbicide resistancemutation in Hra to the S4 mutation indicated that the Hra mutation wasin the same ALS gene as the S4 mutation, namely SURB. Therefore, it wasexpected that the SURB gene isolated from the Hra mutant would be amutant gene, designated SURB-Hra, encoding a herbicide resistant ALS.One phage clone containing the SURB-Hra gene was chosen for furtheranalysis. This phage clone, designated 3, has been deposited at theATCC, Rockville, Md. under accession number ATCC 40237. The phage clonewas digested with Spe I restriction endonuclease to give an 8.3 Kb DNAfragment which was inserted into the Xba I site of plasmid pMuc19, andthe resulting recombinant plasmid, pAGS148, has been deposited at theATCC, Rockville, Md. under accession number ATCC 67124. Plasmids pAGS148and pAGS135 were ligated to each other as described below, and theresulting recombinant plasmid pAGS152 (FIG. 2) was introduced intoAgrobacterium tumefaciens LBA 4404. The resultant Agrobacteriumtumefaciens LBA 4404 (pAGS152) has been deposited at the ATCC, underaccession number ATCC 67126.

A genomic library of DNA from the tobacco C3 mutant was made inbacteriophage lambda and screened for clones which hybridized to thepreviously isolated ALS genes from tobacco. Several phage clones wereisolated and the tobacco DNA inserts were physically mapped withrestriction endonucleases. Two different DNA fragment types,corresponding to the SURA-C3 gene and the SURB gene, were identified.Two phage clones designated 35 and 38, carrying the SURA-C3 gene werechosen for further analysis.

Phage clone 35 was digested with Spe I and Sal I restrictionendonucleases to give the 6.3 kb DNA fragment shown in FIG. 3. This DNAfragment has been inserted into the plasmid vector pUC119 digested withrestriction endonucleases Xba I and Sal I, and the resulting recombinantplasmid, pALS35, has been deposited at the ATCC, Rockville, Md., underaccession number 67424.

In addition to the four tobacco ALS genes, SURA and SURB encoding wildtype herbicide sensitive ALS, and SURA-C3 and SURB-Hra encoding mutantherbicide resistant ALS, ALS genes have been isolated from Arabidopsisthaliana, Beta vulgaris (sugarbeet) and Zea mays (corn). The latter ALSgenes, from herbicide sensitive plants, were obtained from genomic DNAlibraries made in bacteriophage lambda by screening for DNA hybridizingto a previously isolated ALS gene from yeast or tobacco. The wild typeALS gene from sugarbeet was isolated in a phage designated Φ21 andphysically mapped with restriction endonucleases. The DNA fragmentisolated in this phage and two DNA fragments which were subcloned intothe plasmid vector pUC119 are shown in FIG. 7. Plasmid pSBALS216 hasbeen deposited at the ATCC, Rockville, Md. under accession number 67425.

A gene encoding a sulfonylurea resistant form of ALS was also isolatedfrom Arabidopsis. Sulfonylurea resistant mutants of Arabidopsis wereobtained following ethyl methane sulfonate mutagenesis of seeds. Themutant ALS gene was identified from a genomic DNA library made inbacteriophage λ by hybridization to the wild type Arabidopsis gene. A6.1 kb Xba I DNA fragment that contains the entire functional genesubcloned in the plasmid pKAR (FIG. 9) has been deposited at the ATCC,Rockville, Md. under accession number ATCC 67137.

FIG. 1 shows restriction endonuclease maps of DNA fragments containingALS genes isolated from the Hra mutant of tobacco. Based on these maps,two classes of DNA fragments can be distinguished. An approximately 18kilobase nucleic acid insert in phage clone 3 carries the SURB-Hra gene.The insert contains a preferred DNA fragment of the present inventionwhich encodes a herbicide-resistant ALS from tobacco mutant Hra. Thisnucleic acid fragment consists of double-stranded DNA of 8.3±0.5kilobases and has a molecular weight of 5.5±0.3 mega daltons, and has 5'overhang sequences of CTAG at both ends. The 8.3 kilobase nucleic acidfragment between the two Spe I sites hybridized to the ALS gene probeused to screen the genomic library. Restriction endonuclease Spe I canbe used to excise the fragment from the phage using well-knowntechniques.

FIG. 2 shows a physical map of plasmid pAGS152. Plasmids pAGS135 andpAGS148 are not drawn to scale. Restriction endonuclease sites EcoR I(RI), BamH I (B), Xba I (X), Pst I (P), Sal I (S), Spe I (Sp), Nco I(N), Hind III (H), BstE II (Bs), Sma I (Sm), Kpn I (K), Sst I (St), SphI (Sh) and Bgl II (G) are shown. pAGS152 results from the ligation ofthe BamH I- cleaved plasmids pAGS135 (approximately 27 kilobases) andpAGS148 (approximately 12.1 kilobases). Plasmid pAGS135, drawn as acircle, is a wide host range plasmid containing a plant kanamycinresistance gene (NOS:NPT II) and a BamH I cloning site, flanked by theleft border (LB) and the right border (RB) of T-DNA. Plasmid pAGS148,shown as a linear BamH I fragment, consists of the Spe I (Sp) fragment(approximately 8.3 kilobases) of the aspect of the invention (shownflanked by X/Sp and Sp/X), containing the coding sequence for theherbicide-resistant form of ALS, from the Hra mutant, inserted in theXba I site (X) of plasmid pMuc19 (open box). Although Spe I and Xba Irestriction enzymes recognize different sequences, their action resultsin DNA fragments with the same 5' overhanging sequence, viz 5'-CTAG-3'.Thus, Spe I and Xba I digested fragments can be ligated to each other,but the ligation results in a loss of both sites. The hatched box on theinsert fragment corresponds to the coding region of the ALS gene and thearrow denotes the 5'→3' direction of the coding sequence. The nucleicacid fragment is flanked by Hind III, Sph I, Pst I and Sal I sites atone end and by BamH I, Sma I, Kpn I, Sst I and EcoR I sites at the otherend. These enzymes can be used to excise the fragment from the plasmidby complete or partial digestion using well-known techniques. Afterdigestion, the ends of the fragment will be characteristic of theendonuclease used to excise the fragment:

    ______________________________________                                        5' Overhanging Sequence                                                                          3' Overhanging Sequence                                    ______________________________________                                        Spe I     5'-CTAGT-3'  Sph I    5'-C-3'                                                 3'-A-5'               3'-GTACG-5'                                   Hind III  5'-AGCTT-3'  Pst I    5'-G-3'                                                 3'-A-5'               3'-ACGTC-5'                                   Sal I     5'-TCGAC-3'  Kyn I    5'-C-3'                                                 3'-G-5'               3'-CATGG-5'                                   BamH I    5'-GATCC-3'  Sst I    5'-C-3'                                                 3'-G-5'               3'-TCGAG-5'                                   EcoR I    5'-AATTC-3'                                                                   3'-G-5'                                                                       Blunt end                                                           Sma I     5'-GGG-3'                                                                     3'-CCC-5'                                                           ______________________________________                                    

The 8.3 kilobase fragment can be isolated from the restriction digestusing agarose gel electrophoresis. The fragment can be characterized bythe restriction map shown in FIG. 2, and contains the coding sequencefor ALS from mutant plant Hra of Nicotiana tabacum cv. `Xanthi` which isresistant to inhibition by chlorsulfuron and sulfometuron methyl. Thefragment also contains regulatory nucleotide sequences required toexpress the gene in plants.

FIG. 3 shows a restriction endonuclease map of the approximately 6.8 kbpreferred nucleic acid fragment which carries the SURA-C3 gene. This DNAfragment was obtained from lambda phage clone 35 by digestion withrestriction endonucleases SpeI and SalI and was inserted into theplasmid vector pUC119 which had been digested with restrictionendonucleases Xba I and Sal I, as described in the legend to FIG. 2.

FIG. 4 shows a partial nucleotide sequence of a preferred DNA fragmentencoding a herbicide-resistant form of ALS from SURB-Hra gene oftobacco. Nucleotides are indicated by their bases by the followingstandard abbreviations:

A=adenine;

C=cytosine;

T=thymine;

G=guanine.

The beginning of the nucleotide sequence corresponds to Pst I Site (P)885 nucleotide bases preceding the coding sequence, shown on FIG. 2; thesequence ends at base number 2946, which is 67 bases past the end of thecoding sequence shown in FIG. 2. The nucleotide sequence from nucleotideone to nucleotide 884 is believed to contain 5' regulatory sequence(s)required for expression of the encoded ALS. FIG. 4 also shows thededuced amino acid sequence of the ALS protein.

Amino acid residues are indicated by the following abbreviations:

A=alanine;

C=cysteine;

D=aspartic acid;

E=glutamic acid;

F=phenylalanine;

G=glycine;

H=histidine;

I=isoleucine;

K=lysine;

L=leucine;

M=methionine;

N=asparagine;

P=proline;

Q=glutamine;

R=arginine;

S=serine;

T=threonine;

V=valine;

W=tryptophan; and

Y=tyrosine.

The term "amino acids" as used herein is meant to denote theabove-recited natural amino acids and functional equivalents thereof.

FIG. 5 shows a partial nucleotide sequence and its cognate deduced aminoacid sequence, of a preferred DNA fragment encoding aherbicide-resistant form of ALS from the C3 gene of tobacco. Thebeginning of the nucleotide sequence corresponds to the BamH I siteshown in FIG. 3. The coding sequence begins at nucleotide 176 and endsat nucleotide 2175. The nucleotide sequence from nucleotide one tonucleotide 175 is believed to contain 5' regulatory sequence(s)necessary, but not sufficient, for expression of the encoded ALS.Nucleotides and amino acids are indicated by the standard abbreviations,as shown above.

FIG. 6 shows the deduced amino acid sequences of the large subunits ofALS isozymes I, II and III from E. coli (Lines E, F and G respectively),wild type ALS proteins of yeast (Line D), Arabidopsis thaliana (Line C)and Nicotiana tabacum (tobacco) (Lines A and B), encoded by the SURB andSURA genes, respectively. Amino acid residues are indicated by standardabbreviations as shown above. The first amino acid, methionine, of thededuced amino acid sequences of the yeast (line D, FIG. 6) and higherplant (lines A-C) ALS proteins is the putative start of the transitpeptides believed to be involved in translocating the enzymes intomitochondria, in the case of the yeast enzyme, or chloroplasts, in thecase of the plant enzymes. These transit peptides are believed to becleaved off during translocation of the proteins into the organelles andare believed not to be required for ALS activity. The extent of thesetransit peptides is difficult to determine in the absence of data on thein vivo N-termini of the ALS proteins of yeast and higher plants. Basedon the homology with the bacterial ALS proteins the chloroplast andmitochondrial transit sequences may be estimated to extend for 90 aminoacids.

The dotted lines in the sequences are spacing marks inserted to bestalign regions of homology. Vertical lines highlight the amino acidresidues that are conserved between adjacent sequences of FIG. 6. Thehomology between tobacco and Arabidopsis ALS proteins (lines A to C),which derive from two different plant families, is striking. Even moreunexpected, considering the evolutionary distance between microbes andhigher plants, is the finding that the amino acid residues which areconserved between the bacterial (lines E to G) and the yeast (line D)ALS proteins are largely conserved between these proteins and the plantALS proteins.

FIG. 7 shows a restriction endonuclease map of the approximately 17.5kilobase nucleic acid insert in phage clone φ21 carrying the sugarbeetALS gene. Two smaller DNA fragments which also contain the sugarbeet ALSgene and which were subcloned into the pUC119 plasmid vector are alsoshown.

FIG. 8 shows deduced amino acid sequences of wild type ALS proteins fromthe plants Nicotiana tabacum (tobacco) (Lines A and B), Arabidopsisthaliana (Line C) Beta vulgaris cv. sennica (sugarbeet) (Line D) and apartial sequence of the ALS protein from maize (Line E). The dottedlines in the sequences are spacing marks to best align regions ofhomology. Vertical lines highlight the amino acid sequences that areconserved between adjacent sequences. The homology between all of theplant ALS proteins is very extensive. Based upon this, a mutation in oneplant ALS gene causing an amino acid substitution that results insulfonylurea herbicide resistant ALS would be expected to have ananalogous effect if it were present in any other plant ALS gene.

FIG. 9 shows a diagram of plasmid pKAR. Restriction sites Eco RI (RI),Cla I (C), Hind III (H), Bam HI (B), Sal I (S), Xba I (X), and Nco I (N)are shown. The numbers between the restriction sites are the distancesbetween the sites, in kilobases. The open box represents the chimericNOS:NPT II gene in which the coding sequence of a neomycinphosphotransferase (NPT II) gene on a 1 kilobase Hind III-Bam HIfragment, is fused to the promoter region of a nopaline synthase (NOS)gene, on a 0.65 kilobase Cla I-Hind III fragment, and to the 3'regulatory sequences of the NOS gene on a 0.7 kilobase Bam HI-Cla Ifragment. The NOS:NPT II chimeric gene expresses kanamycin resistance inplant cells. The hatched box represents 1.3 kilobases of a bacterialgene for NPT I that expresses kanamycin resistance in E. coli andAgrobacterium. The stippled box represents a 5.3 kilobase Xba I fragmentcontaining the coding sequence of the herbicide-resistant ALS fromArabidopsis.

FIG. 10 shows a partial nucleotide sequence and its cognate deducedamino acid sequence, of a preferred DNA fragment encoding aherbicide-resistant form of ALS. The coding sequence begins atnucleotide 506 and ends at nucleotide 2518. The nucleotide sequence fromnucleotide one to nucleotide 505 is believed to contain the 5'regulatory sequence(s) required for expression of the encoded ALS.Nucleotides and amino acids are indicated by the standard abbreviations,as shown above.

The amino acid residues which are conserved in all of the ALS sequencesin FIG. 6 are believed to be important for the binding of substrates,herbicides, coenzymes, etc. These sequences are believed to besubstantially conserved in all ALS proteins. The residues which arepartially conserved in the different ALS proteins may participate inless conserved aspects of enzyme function, such as those which governits herbicide sensitivity and its end-product inhibition. Examples ofthis would include the resistance of bacterial isozyme I to sulfometuronmethyl and chlorsulfuron, and of bacterial isozyme II to end-productinhibition by valine. Finally, those residues which are not conservedbetween the proteins probably reside in the framework of the ALS proteinwhere sequence divergence is less disruptive to enzyme function.

Although not wishing to be bound by theory, binding of sulfonylureaherbicides to ALS during acetolactate synthesis is believed to befacilitated by the binding of a first pyruvate molecule to the enzyme.However, the binding of a sulfonylurea herbicide molecule is competitivewith the binding of a second pyruvate molecule to the enzyme.Sulfonylurea herbicide sensitivity is conserved through evolution inmost ALS enzymes. From these facts, it was deduced that the binding ofthe sulfonylurea herbicide occurs at or proximal to one or more of theconserved amino acids in the ALS proteins. In fact, Applicant hasdiscovered that substitutions for one or more of 10 specific amino acidresidues in one or more of the 7 substantially conserved sub-sequences Athrough G will confer herbicide resistance and are claimed. It isexpected that substitution at other amino acid residues in thesubstantially conserved sub-sequences will also confer herbicideresistance.

Sulfonylurea herbicide resistance in bacteria, yeast and higher plants,which resistance cosegregates with herbicide-resistant forms of ALS,results from mutations in the structural genes for ALS. Comparing thenucleotide sequences of ALS genes of organisms encoding herbicidesensitive and herbicide-resistant forms of ALS allows one to determinewhich amino acid residues are important for herbicide inhibition of theenzyme. One mutation in the E. coli ilvG gene, which results in anenzyme with increased resistance to sulfometuron methyl inhibition, andwith reduced catalytic activity, was determined to result in analanine-to-valine substitution at position 122 (FIG. 6). Anothersulfometuron methyl resistance mutation in this gene was determined toresult in a alanine-to-serine substitution at the same position. Thisalanine residue is conserved in all ALS enzymes except bacterial isozymeI (FIG. 6), which is naturally resistant.

Many genes encoding herbicide-resistant ALS enzymes have been isolatedfrom spontaneous sulfonylurea-resistant yeast mutants. Sequencing ofthese genes has shown the molecular basis of resistance to be basechanges which result in amino acid substitutions at ten differentpositions in the protein (Table 2), residues 121, 122, 197, 205, 256,359, 384, 588, 591 and 595 (numbering relative to the positions in FIG.6).

                  TABLE 2                                                         ______________________________________                                        Spontaneous Mutations of the Yeast ALS Gene                                   Resulting in Sulfonylurea Herbicide Resistance                                Amino Acid                                                                             Wild Type Wild Type  Mutant                                                                              Amino Acid                                Positions                                                                              Codon     Amino Acid Codon Substitution                              ______________________________________                                        121      GGT       Gly        AGT   Ser                                       122      GCT       Ala        CCT   Pro                                                                     GAT   Asp                                                                     GTT   Val                                                                     ACT   Thr                                       197      CCA       Pro        TCA   Ser                                                                     CGA   Arg                                       205      GCT       Ala        GAT   Asp                                                                     ACT   Thr                                       256      AAG       Lys        GAG   Glu                                                                     ACG   Thr                                                                     AAC   Asn                                       359      ATG       Met        GTG   Val                                       384      CAC       Asp        GAA   Glu                                                                     GTC   Val                                                                     AAC   Asn                                       588      GTT       Val        GCT   Ala                                       591      TGG       Trp        CGG   Arg                                                                     AGG   Arg                                                                     TGT   Cys                                                                     TGC   Cys                                                                     GGG   Gly                                                                     TTG   Leu                                                                     TCG   Ser                                                                     GCG   Ala                                       595      TTC       Phe        TTA   Leu                                       ______________________________________                                    

At six of these positions, 122, 197, 205, 256, 384 and 591 (Table 2),more than one substitution that confers herbicide resistance has beenobtained. At position 122, at which an alanine residue is present in allknown wild type ALS enzymes except E. coli isozyme I, substitutions ofaspartic acid, proline, threonine or valine result insulfonylurea-resistant ALS. At position 197, at which a proline residueis present in all known wild-type ALS enzymes except E. coli isozymes IIand III, substitutions of serine or arginine result insulfonylurea-resistant ALS. At position 205, at which an alanine residueis present in all known wild type ALS enzymes, substitutions of asparticacid or threonine result in sulfonylurea-resistant ALS. At position 256,at which a lysine residue is present in all known wild type ALS enzymes,substitutions of glutamic acid, asparagine or threonine result insulfonylurea-resistant ALS. At position 384 at which an aspartic acid ispresent in all known wild type ALS enzymes, substitutions of glutamicacid, asparagine or valine result in sulfonylurea-resistant ALS. Atposition 591, at which a tryptophan is present in all known wild typeALS enzymes except E. coli isozyme I, substitutions of alanine,cysteine, glycine, leucine, arginine or serine result insulfonylurea-resistant ALS.

Mutants resistant to sulfonylurea herbicides resulting from single aminoacid substitutions at the other four positions, 121, 359, 588 and 595,have been obtained. At position 121, at which glycine is present in allknown ALS enzymes, substitution of serine results in asulfonylurea-resistant ALS. At position 359, at which methionine ispresent in all known ALS enzymes, substitution of valine results in asulfonylurea-resistant ALS. At position 588, at which valine is presentin all known ALS enzymes, substitution of alanine results insulfonylurea-resistant ALS. At position 595, at which phenylalanine ispresent in all known ALS enzymes except E. coli isozyme III,substitution of leucine results in sulfonylurea-resistant ALS.

Oligonucleotide-directed site specific mutations, which result in aminoacid substitutions at positions 121, 122, 197, 205, 256, 359, 384, 588,591, and 595 have been made in the yeast gene encoding ALS (Table 3).

                  TABLE 3                                                         ______________________________________                                        Site-Directed Mutations of the Yeast ALS Gene                                 Resulting in Sulfonylurea Herbicide Resistance                                Amino Acid                                                                             Wild Type Wild Type  Mutant                                                                              Amino Acid                                Positions                                                                              Codon     Amino Acid Codon Substitution                              ______________________________________                                        121      GGT       Gly        AAT   Asn                                                                     GCT   Ala                                                                     GAT   Asp                                       122      GCT       Ala        TCT   Ser                                                                     GTT   Val                                                                     ACT   Thr                                                                     CCT   Pro                                                                     AAT   Asn                                                                     ATT   Ile                                                                     CAT   His                                                                     CGT   Arg                                                                     CTT   Leu                                                                     TAT   Tyr                                                                     TGT   Cys                                                                     TTT   Phe                                                                     GAA   Glu                                                                     ATG   Met                                                                     AAA   Lys                                                                     CAA   Gln                                                                     TGG   Trp                                       197      CCA       Pro        CAA   Gln                                                                     GAA   Glu                                                                     GCA   Ala                                                                     GGT   Gly                                                                     TGG   Trp                                                                     TAC   Tyr                                                                     TGC   Cys                                                                     GTT   Val                                       205      GCT       Ala        CGT   Arg                                                                     TGT   Cys                                                                     GAA   Glu                                                                     TGG   Trp                                                                     TAT   Tyr                                                                     GTT   Val                                                                     AAT   Asn                                       256      AAG       Lys        GAC   Asp                                                                     CCG   Pro                                       359      ATG       Met        CCA   Pro                                                                     GAG   Glu                                                                     CAA   Gln                                                                     AAG   Lys                                                                     TAT   Tyr                                                                     TGT   Cys                                       384      GAC       Asp        CCA   Pro                                                                     TGG   Trp                                                                     TCC   Ser                                                                     GGT   Gly                                                                     TGC   Cys                                       583      GTT       Val        AGT   Ser                                                                     AAT   Asn                                                                     TGG   Trp                                                                     TGT   Cys                                       591      TGG       Trp        GAC   Asp                                                                     GAG   Glu                                                                     TTC   Phe                                                                     CAC   His                                                                     TAC   Tyr                                                                     ATA   Ile                                                                     GTG   Val                                                                     AAG   Lys                                                                     ATG   Met                                                                     AAC   Asn                                                                     CAG   Gln                                                                     ACG   Thr                                       595      TTC       Phe        GGT   Gly                                                                     AAC   Asn                                                                     CGC   Arg                                                                     TGC   Cys                                                                     CCA   Pro                                                                     TCC   Ser                                                                     TGG   Trp                                       ______________________________________                                    

At position 122, mutations resulting in eighteen amino acidsubstitutions for alanine, which is present in wild type ALS, have beenmade. The nineteenth substitution (aspartic acid) was isolatedpreviously as a spontaneous mutation and was therefore not remade. Eachsubstitution, except for glycine, results in sulfonylurea-resistant ALS.At position 205, mutations resulting in substitutions for alanine, thewild type residue, of cysteine, glutamic acid, arginine, tryptophan,tyrosine, valine or asparagine result in sulfonylurea-resistant ALS. Atposition 256, mutations resulting in substitutions for lysine, the wildtype residue, of aspartic acid or proline result insulfonylurea-resistant ALS. At position 359, mutations resulting insubstitutions for methionine, the wild type residue, of glutamic acid,glutamine, lysine, tyrosine, cysteine or proline result insulfonylurea-resistant ALS. At position 384, mutations resulting inamino acid substitutions for aspartic acid, the wild type residue, ofcysteine, glycine, proline, serine or tryptophan result insulfonylurea-resistant ALS. At position 591, mutations resulting inamino acid substitutions for tryptophan, the wild type residue, ofaspartic acid, glutamic acid, phenylalanine, histidine, isoleucine,lysine, valine, methionine, asparagine, glutamine, threonine or tyrosineresult in sulfonylurea-resistant ALS. At position 121, mutationsresulting in substitutions for glycine, the wild type residue, ofasparagine, alanine or aspartic acid result in sulfonylurea-resistantALS. At position 197, mutations resulting in substitutions for proline,the wild type residue, of glutamine, glutamic acid, alanine, glycine,tryptophan, tyrosine, cysteine or valine result insulfonylurea-resistant ALS. At position 583, mutations resulting insubstitutions for valine, the wild type residue, of serine, asparagine,tryptophan or cysteine result in sulfonylurea-resistant ALS. At position595, mutations resulting in substitutions for phenylalanine, the wildtype residue, of glycine, asparagine, arginine, cysteine, proline,serine or tryptophan result in sulfonylurea-resistant ALS.

All mutations described in Tables 2 and 3 resulted in enzymes which wereactive and less inhibited by sulfonylurea herbicides than the wild type.Taken in total these results indicate that most substitutions at these10 positions result in enzymatically active herbicide resistant ALS.

The deduced amino acid sequences of the wild type ALS proteins fromtobacco, Arabidopsis, sugarbeet and corn (partial) are shown in FIG. 7.The amino acid residues at positions 121, 122, 197, 205, 256, 359, 384,588, 591 and 595 (numbering of positions from FIG. 6) in all the plantenzymes are the same as those present in the wild type herbicidesensitive yeast protein (FIG. 6). The deduced amino acid sequence of thetobacco ALS gene SURB-Hra, which encodes a herbicide-resistant ALS, isshown in FIG. 4. The mutant gene of FIG. 4 was derived from a tissueculture line which had undergone two successive spontaneous mutations.The two mutations have been shown to be genetically linked, andintroduction of this fragment into sensitive tobacco cells confers uponthe cells the same level of herbicide resistance as is found for theoriginal highly resistant mutant tobacco plant from which the fragmentwas derived. Based on these facts, it was expected that there would betwo amino acid substitutions in the enzyme encoded by the fragment. Acomparison of the deduced amino acid sequence of the mutant ALS with thededuced amino acid sequence of the wild type ALS reveals that the mutantALS has a proline-to-alanine substitution at position 197 (FIG. 6) and atryptophan-to-leucine substitution at position 591 (FIG. 6). Based onthe foregoing, it was determined that substitutions at proline 197 andtryptophan 591 residues confer herbicide resistance. The deduced aminoacid sequence of a second mutant tobacco ALS gene, SURA-C3, whichencodes a sulfonylurea herbicide resistant ALS, is shown in FIG. 5. Acomparison of the deduced amino acid sequences of the mutant and wildtype ALS enzymes (FIG. 5 and FIG. 6, line B) reveals that the mutant ALShas a single substitution, proline-to-glutamine, at position 197. The C3cell line from which the SURA-C3 gene was obtained showed selectiveherbicide resistance. That is, the C3 mutation conferred resistance tothe sulfonylurea herbicides chlorsulfuron and sulfometuron methyl, butnot to an imidazolinone herbicide.

The deduced amino acid sequence of a mutant Arabidopsis gene whichencodes a sulfonylurea herbicide resistant ALS, is shown in FIG. 10. Acomparison of the deduced amino acid sequences of the mutant and wildtype ALS enzymes (FIG. 10 and FIG. 6, line C) reveals that the mutantALS has a single substitution, proline-to-serine, at position 197. Thismutation, like the C3 mutation, conferred resistance to the sulfonylureaherbicides chlorsulfuron and sulfometuron methyl, but not to animidazolinone herbicide. Thus, two different amino acid substitutionsfor proline at position 197 result in selective herbicide resistance.

The identification of amino acid substitutions in herbicide-resistantALS enzymes from plants at positions 197, from the C3, Hra andArabidopsis mutants, and 591 from the Hra mutant, indicates thatsubstitutions at positions operable in yeast ALS are also operable inplant ALS.

While the amino acid residue present at positions 121, 122, 197, 205,256, 359, 384, 588, 591 and 595 are conserved in all wild type herbicidesensitive ALS enzymes so far characterized from eucaryotes, somesubstitutions at these positions are found in wild type bacterial ALSenzymes. E. coli isozyme I has a serine rather than alanine at position122 and a glutamine rather than tryptophan at position 591, E. coliisozyme II has a serine rather than proline at position 197 and E. coliisozyme III has an alanine rather than proline at position 197 and anisoleucine rather than phenylalanine at position 595. Each of these E.coli ALS isozymes is more resistant (from 50-fold to greater than10,000-fold) to inhibition by (particular) sulfonylurea herbicides thanplant or yeast ALS. Furthermore, a site-directed mutation causing aserine-to-proline substitution at position 197 in E. coli ALS IIrendered the mutant enzyme 100 fold more sensitive to inhibition, i.e.,as sensitive as wild type higher plant enzymes. Thus, proline atposition 197 is involved in herbicide binding in E. coli ALS II as wellas in yeast and higher plant ALS.

In addition, site-directed mutations which result intryptophan-to-leucine and glutamine-to-tryptophan substitutions atposition 591 in ALS II and ALS I, respectively, of E. coli have beenmade. The mutation in ALS II makes the enzyme more herbicide resistantthan the wild type ALS II, while the mutation in ALS I makes it moresensitive than wild type ALS I.

The site-directed mutations at positions 197 and 591 in ALS I and ALS IIof E. coli affect inhibition by herbicide of the mutant enzymes in amanner predicted from the herbicide-resistant mutant yeast and plant ALSproteins. These experimental findings support the universality of theamino acid residues involved in herbicide binding to ALS enzymes fromdiverse sources.

Characterization of Nucleic Acid Fragments Encoding Herbicide-ResistantALS

According to the present invention, the amino acid residues of ALS thatcorrespond to ε₁ and ε₂ in amino acid sub-sequence A, α₁ in amino acidsub-sequence B, δ₂ in amino acid sub-sequence C, γ₁ in amino acidsub-sequence D, λ₁ in amino acid sub-sequence E, β₃, β₇ and β₈ in aminoacid sub-sequence F and σ₁ in amino acid sub-sequence G of FIG. 6(referred to hereinafter as positions 122, 121, 197, 205, 256, 384, 591,595, 588 and 359 respectively) are important in herbicide sensitivity orresistance of ALS enzymes regardless of the biological source of theseenzymes, and any nucleotide sequence encoding a plant ALS can be alteredto direct synthesis of a herbicide-resistant ALS by virtue of amino acidsubstitutions at these residues. The nucleic acid fragment of thepresent invention is characterized in that at least one of the followingconditions is met:

a) The nucleic acid fragment encodes an amino acid other than glycine atposition 121. Preferably the amino acid is serine, alanine, asparagine,glutamine, glutamic acid, threonine, or aspartic acid. Most preferablythe amino acid is serine, asparagine, alanine or aspartic acid.

b) The nucleic acid fragment encodes an amino acid other than alanine atposition 122. Most preferably, the amino acid is any other than glycine.

c) The nucleic acid fragment encodes an amino acid other than proline atposition 197. Preferably, the amino acid is alanine, glycine, arginine,tyrosine, tryptophan, serine, valine, cysteine, glutamine, or glutamicacid. Most preferably the amino acid is alanine, serine, arginine,glutamine, glutamic acid, tryptophan or tyrosine.

d) The nucleic acid fragment encodes an amino acid other than alanine atposition 205. Preferably, the amino acid is any other than glycine orproline. Most preferably, the amino acid is threonine, cysteine,aspartic acid, glutamic acid, tryptophan, arginine, valine, asparagineor tyrosine.

e) The nucleic acid fragment encodes an amino acid other than lysine atposition 256. Preferably, the amino acid is threonine, serine, glutamicacid, aspartic acid, proline, asparagine or glutamine. Most preferably,the amino acid is threonine, glutamic acid, aspartic acid, proline orasparagine.

f) The nucleic acid fragment encodes an amino acid other than methionineat position 359. Preferably, the amino acid is glutamic acid, glutamine,asparagine, aspartic acid, proline, valine, leucine, isoleucine, lysine,arginine, tyrosine, phenylalanine or cysteine. Most preferably the aminoacid is glutamic acid, proline, glutamine, lysine, tyrosine, cysteine orvaline.

g) The nucleic acid fragment encodes an amino acid other than asparticacid at position 384. Preferably, the amino acid is glycine, alanine,valine, leucine, isoleucine, serine, threonine, cysteine, glutamic acid,proline, asparagine, glutamine, tryptophan, or histidine. Mostpreferably, the amino acid is glycine, valine, serine, cysteine,glutamic acid, proline, asparagine or tryptophan.

h) The nucleic acid fragment encodes an amino acid other than valine atposition 588. Preferably the amino acid is alanine, serine, threonine,asparagine, glutamine, tryptophan, histidine, cysteine or methionine.Most preferably the amino acid is alanine, serine, asparagine,tryptophan or cysteine.

i) The nucleic acid fragment encodes an amino acid other than tryptophanat position 591. Most preferably, the amino acid is other than proline.

j) The nucleic acid fragment encodes an amino acid other thanphenylalanine at position 595. Preferably, the amino acid is any otherthan tyrosine, aspartic acid or glutamic acid. Most preferably the aminoacid is leucine, glycine, proline, serine, asparagine, arginine,tryptophan or cysteine.

In one embodiment, position 121 resides within amino acid sub-sequence Aas follows:

    PGε.sub.2 A

wherein P, G and A are as defined above. To confer herbicide resistance,ε₂ is an amino acid other than glycine. Most preferably ε₂ is the aminoacid serine, alanine, asparagine or aspartic acid. This sub-sequencebegins about 24 residues from the beginning of a substantially conservedamino acid sequence

    HEQ,

i.e.,

    PGε.sub.2 A . . . HEQ.

In one embodiment, position 122 resides within amino acid sub-sequence Aas follows:

    PGGε.sub.1

wherein P and G are as defined above. To confer herbicide resistance ε₁is a natural amino acid other than alanine. Most preferably ε₁ is anyamino acid except glycine. This sub-sequence begins about 24 residuesfrom the beginning of a substantially conserved amino acid sequence

    HEQ,

i.e.,

    PGGε.sub.1. . . HEQ.

In one embodiment, position 197 resides within amino acid sub-sequence Bas follows:

    GQVα.sub.1

wherein G, Q, and V are as defined above. To confer herbicide resistanceα1 is an amino acid other than proline. Most preferably, α₁ is alanine,glycine, tyrosine, tryptophan, valine, cysteine, glutamic acid,arginine, serine or glutamine. This sub-sequence begins about 20residues from the end of one substantially conserved amino acid sequence

    SGPGATN

and about 55 residues from the beginning of a second substantiallyconserved amino acid sequence

    SGRPGP,

i.e.,

    SGPGATN . . . GQVα.sub.1 . . . SGRPGP.

In one embodiment, position 205 resides within an amino acidsub-sequence C as follows:

    IGδ.sub.1 Dδ.sub.2 FQE

wherein I, G, D, F, Q, and E are as defined above, δ₁ represents anamino acid residue which can vary according to the source of the enzyme,but is most commonly T. To confer herbicide resistance δ₂ is an aminoacid other than alanine. Most preferably, δ₂ is threonine, cysteine,aspartic acid, glutamic acid, arginine, valine, asparagine, tyrosine ortryptophan. This sub-sequence begins about 5 residues from the end of asubstantially conserved amino acid sequence

    GQV

and about 43 residues from the beginning of a second substantiallyconserved amino acid sequence

    SGRPGP,

i.e.,

    GQV . . . IGδ.sub.1 Dδ.sub.2 FQE . . . SGRPGP.

In one embodiment, position 256 resides within an amino acidsub-sequence D as follows:

    Pλ.sub.1 D

wherein P and D are as defined above. To confer herbicide resistance λ₁is an amino acid other than lysine. Most preferably, λ₁, is threonine,glutamic acid, aspartic acid, asparagine or proline. This sub-sequence Dbegins about 6 residues from the end of a substantially conserved aminoacid sequence

    SGRPGP

i.e.,

    SGRPGP . . . pλ.sub.1 D.

In one embodiment, position 359 resides within an amino acidsub-sequence G as follows

    MLGσ.sub.1 HG

wherein M, L, G and H are defined as above. To confer herbicideresistance, σ₁ is an amino acid other than methionine. Most preferably,σ₁ is proline, glutamine, lysine, tyrosine, cysteine, glutamic acid orvaline. This sub-sequence ends about 20 residues from the beginning of asubstantially conserved amino acid sequence

    RFDDR

i.e.,

    MLGσ.sub.1 HG . . . RFDDR.

In one embodiment, position 384 resides within an amino acidsub-sequence G as follows

    RFDγ.sub.1 R

wherein R, F, and D are as defined above. To confer herbicideresistance, γ₁ is an amino acid other than aspartic acid. Mostpreferably, γ₁ is glycine, valine, cysteine, serine, glutamic acid,proline, asparagine or tryptophan.

This sub-sequence begins about 20 residues from the end of asubstantially conserved amino acid sequence

    MLGMHG,

i.e.,

    MLGMHG . . . RFDγ.sub.1 R.

In one embodiment, position 588 resides within an amino acidsub-sequence F as follows

    Gβ.sub.1 β.sub.8 β.sub.2 Qβ.sub.3 β.sub.4 β.sub.5 β.sub.6 β.sub.7

wherein G and Q are defined above, β₁ to β₈ will vary depending upon-thesource of the enzyme. β₁ is usually methionine, β₃ is usually tryptophanand β₇ is usually phenylalanine. To confer herbicide resistance β₈ is anamino acid other than valine. Most preferably β₈ is alanine, serine,asparagine, tryptophan or cysteine. This sub-sequence begins about 49residues from the end of a substantially conserved amino acid sequence

    GLPAA

i.e.,

    GLPAA . . . Gβ.sub.1 β.sub.8 β.sub.2 Qβ.sub.3 β.sub.4 β.sub.5 β.sub.6 β.sub.7.

In one embodiment, position 591 resides within an amino acidsub-sequence F as follows

    Gβ.sub.1 Vβ.sub.2 Qβ.sub.3 β.sub.4 β.sub.5 β.sub.6 β.sub.7

wherein G, V and Q are defined above, β₁ to β₇ will vary depending uponthe source of the enzyme. β₁ is usually methionine and β₇ is usuallyphenylalanine. To confer herbicide resistance β₃ is any amino acid otherthan tryptophan. Most preferably β₃ is any amino acid other thanproline. This sub-sequence begins about 49 residues from the end ofanother substantially conserved amino acid sequence

    GLPAA,

i.e.,

    GLPAA . . . Gβ.sub.1 Vβ.sub.2 qβ.sub.3 β.sub.4 β.sub.5 β.sub.6 β.sub.7.

In one embodiment, position 595 resides within an amino acidsub-sequence F as follows

    Gβ.sub.1 Vβ.sub.2 Qβ.sub.3 β.sub.4 β.sub.5 β.sub.6 β.sub.7

wherein G. V and Q are defined above. β₁ to β₇ will vary depending uponthe source of the enzyme. β₁ is usually methionine and β₃ is usuallytryptophan. To confer herbicide resistance, β₇ is an amino acid otherthan phenylalanine. Most preferably, β₇ is leucine, glycine, proline,serine, asparagine, arginine, tryptophan or cysteine. This sub-sequencebegins about 49 amino acids from the end of a substantially conservedamino acid sequence

    GLPAA

i.e.,

    GLPAA . . . Gβ.sub.1 Vβ.sub.2 Qβ.sub.3 β.sub.4 β.sub.5 β.sub.6 β.sub.7.

Herbicide resistance can be achieved by any one of the above describedamino acid substitutions and by combinations thereof.

The precise amino acid substitutions required for herbicide resistancecan be achieved by mutating a nucleic acid fragment encoding a herbicidesensitive ALS from any plant of interest generally as follows:

(1) isolate genomic DNA or mRNA from the plant;

(2) prepare a genomic library from the isolated DNA or a cDNA libraryfrom the isolated RNA;

(3) identify those phages or plasmids which contain a DNA fragmentencoding ALS;

(4) sequence the fragment encoding the ALS;

(5) sub-clone the DNA fragment carrying the ALS gene into a cloningvehicle which is capable of producing single-stranded DNA:

(6) synthesize an oligonucleotide of about 15 to 20 nucleotides which iscomplementary to a particular ALS nucleotide sequence encoding one ofthe amino acid sub-sequences recited above except for the nucleotidechange(s) required to direct a mutation to a codon for an amino acidselected for its ability to confer herbicide resistance;

(7) anneal the oligonucleotide to the single-stranded DNA containing theregion to be mutated and use it to prime synthesis in vitro of acomplementary DNA strand forming a heteroduplex;

(8) transform bacterial cells with the heteroduplex DNA;

(9) screen the transformed bacterial cells for those cells which containthe mutated DNA fragment by a) immobilizing the DNA on a nitrocellulosefilter, b) hybridizing it to the 5'-³² P labelled mutagenicoligonucleotide at ambient temperature, and c) washing it underconditions of increasing temperature so as to selectively dissociate theprobe from the wild-type gene but not the mutant gene;

(10) isolate a double-stranded DNA fragment containing the mutation fromthe cells carrying the mutant gene; and

(11) confirm the presence of the mutation by DNA sequence analysis.

An amino acid substitution required for herbicide resistance can also beachieved by substituting a nucleotide sequence of a plant ALS gene whichencodes a sequence of amino acids containing the amino acid to besubstituted with another nucleotide sequence, which encodes thecorresponding stretch of amino acids containing the desiredsubstitution, derived from any natural ALS gene (including microbial) orfrom a synthetic source.

Preparation of Herbicide-Resistant Plants

The nucleic acid fragments of the present invention can be used tointroduce herbicide resistance into plants. In order to introduce anucleic acid fragment which includes a gene encoding herbicide resistantALS into different plants, a wide variety of techniques are useddepending on the species or cultivar desired. In general, explants orprotoplasts can be taken or produced from either in vitro or soil grownplants. Explants or protoplasts may be produced from cotyledons, stems,petioles, leaves, roots, immature embryos, hypocotyls, inflorescences,etc. In theory, any tissue which can be manipulated in vitro to giverise to new callus or organized tissue growth can be used for genetictransformation.

To achieve transformation, explants or protoplasts may be coculturedwith Agrobacterium, which can be induced to transfer nucleic acidfragments located between the T-DNA borders of the Ti plasmid to theplant cells. Another method, less commonly used, is direct DNA uptake byplant protoplasts. With this method, the use of Agrobacterium isbypassed and DNA is taken up directly by the protoplasts under theappropriate conditions.

In the examples, a variety of explants from different plants have beencocultured with Agrobacterium to achieve transformation to herbicideresistance. These explants were cultured to permit callus growth. Thecallus was then tested directly for resistance to sulfonylureas, orplants were regenerated and the plants were tested for sulfonylurearesistance. Testing consisted of an enzyme assay of plant cell extractsfor the presence of ALS activity resistant to herbicide and/or growth ofplant cells in culture or of whole plants in the presence of normallyinhibitory concentrations of herbicide.

The DNA fragments are comprised of a region coding for the synthesis ofherbicide-resistant ALS and a region providing for expression of thecoding sequence in plants. The 8.3 kb DNA fragment shown in FIG. 2 whichcodes for the herbicide-resistant ALS protein shown in FIG. 4 containsabout 800 bp in the 5' direction (upstream) of the coding region,sufficient for expression of the protein in plants. This DNA fragmentcan confer resistance to chlorsulfuron up to 2000 ppb in transformedtobacco calluses. Plants regenerated from the transformed cells alsoshow resistance at the whole plant level. The 6.3 kb DNA fragment shownin FIG. 3 which codes for the herbicide resistant ALS protein shown inFIG. 5 contains 2.5 kb in the 5' direction (upstream) and 1.8 kb in the3' direction (downstream) of the coding region sufficient for expressionof the protein in plants. This DNA fragment can confer resistance tochlorsulfuron at 2ppb in transformed tobacco calluses.

The 5.3 kb DNA fragment shown in FIG. 9, which codes for the herbicideresistant ALS protein shown in FIG. 10, contains about 2.5 kb in the 5'direction (upstream) and about 0.8 kb in the 3' direction (downstream)of the coding region sufficient for expression of the protein in plants.This DNA fragment can confer resistance to chlorsulfuron at 30 ppb intransformed tobacco calluses.

In work which is on-going, DNA fragments containing site-directedmutations in the SURA gene that are expected to code for herbicideresistant ALS have been made. These mutations result in the followingamino acid substitutions: Ala 122 to Ser, known to be operable in E.coli ALS isozyme II and yeast ALS, Ala 122 to Val, known to be operablein E. coli ALS isozyme II and yeast ALS, Ala 122 to Pro, known to beoperable in yeast ALS, Pro 197 to Ser, known to be operable in yeastALS, and E. coli ALS II enzyme, Pro 197 to Ala, known to be operable inALS encoded by the SURB-Hra gene of tobacco, Ala 205 to Asp, known to beoperable in yeast ALS, Lys 256 to Glu, known to be operable in yeastALS, Asp 384 to Val, known to be operable in yeast ALS and Trp 591 toLeu, known to be operable in yeast ALS and ALS encoded by the SURB-Hragene of tobacco. By combining the above mutations, double mutations,resulting in two amino acid substitutions such as Ala 122 to Ser and Pro197 to Ser, or Ala 122 to Set and Pro 197 to Ala, or Pro 197 to Ala andTrp 591 to Leu, or Pro 197 to Ser and Trp 591 to Leu have also beenmade. These mutations were made in a DNA fragment that included onlyabout 180 bp in the 5' direction (upstream) and only about 600 bp in the3' direction (downstream) of the ALS coding sequence. These DNAfragments were introduced into tobacco by transformation. Herbicideresistance was not expressed in these transformants. The lack ofupstream DNA sequences necessary for expression of the mutant ALS geneswas thought-to be the reason for this. DNA fragments containing severalof these in vitro constructed mutations have been inserted into an SURBgene fragment that contains the upstream and downstream DNA sequencesnecessary for expression. These recombinant DNA fragments wereintroduced into tobacco by transformation. In this way, the single aminoacid substitutions Pro 197 to Set, Pro 197 to Ala, Ala 205 to Asp, Trp591 to Leu, and the double amino acid substitutions Pro 197 to Ala, Trp591 to Leu, and Pro 197 to Ser, Trp 591 to Leu have thus fax been shownto confer herbicide resistance in tobacco tissue culture cells. The Ala205 to Asp mutation had not before been seen in a plant gene encodingherbicide resistant ALS and therefore further demonstrates thatsubstitutions found to be operable in yeast ALS are also operable inplant ALS. The in vitro constructed Pro 197 to Set and Pro 197 to Alamutations both confer resistance to the sulfonylurea herbicidechlorsulfuron without affecting sensitivity to an imidazolinone. Thus,three different amino acid substitutions for Pro 197, Set, Ala and Gln(found in the SURA-C3 encoded protein) confer selective herbicideresistance, suggesting that any substitution at this position will dolikewise.

Site directed mutations that are expected to code for herbicideresistant ALS have also been made in the sugarbeet ALS gene. Thesemutations result in the following amino acid substitutions: Ala 122 toPro, known to be operable in yeast ALS, Pro 197 to Ala, known to beoperable in ALS encoded by the SURB-Hra gene of tobacco, Trp 591 to Leu,known to be operable in yeast ALS and in ALS encoded by the SURB-Hragene of tobacco and the double mutant, Pro 197 to Ala and Trp 591 toLeu, known to be operable in ALS encoded by the SURB-Hra gene oftobacco. DNA fragments carrying these mutant sugarbeet ALS genes havebeen introduced into tobacco and sugarbeet cells by transformation. ThePro 197 to Ala and Trp 591 to Leu single amino acid substitutions andthe double substitution of Pro 197 to Ala and Trp 591 to Leu conferredresistance to chlorsulfuron in both tobacco and sugarbeets. ALS genecarrying the Ala 122 to Pro substitution did not yield chlorsulfuronresistant transformants. This amino acid substitution has been shown toconfer selective herbicide resistance when present in yeast ALS. Whilethe substitution results in resistance to sulfometuron methyl, themutant enzyme remains sensitive to chlorsulfuron. Thus, it would not beexpected to yield chlorsulfuron resistance when present in plant ALS.This type of selective resistance gene represents a particularly usefulmanifestation of the invention.

The nucleic acid fragments of the invention generally can be introducedinto plants directly or in a nucleic acid construct comprising thedesired nucleic acid fragment. The nucleic acid construct can be derivedfrom a bacterial plasmid or phage, from the Ti- or Ri-plasmids, from aplant virus or from an autonomously replicating sequence. One preferredmeans of introducing the nucleic acid fragment into plant cellscomprises use of Agrobacterium tumefaciens containing the nucleic acidfragment between T-DNA borders either on a disarmed Ti-plasmid (that is,a Ti-plasmid from which the genes for tumorigenicity have been deleted)or in a binary vector in trans to a Ti-plasmid with Vir functions. TheAgrobacterium can be used to transform plants by inoculation of tissueexplants, such as stems or leaf discs, or by co-cultivation with plantprotoplasts. Another preferred means of introducing the present nucleicacid fragment comprises direct introduction of the fragment or a vectorcontaining the fragment into plant protoplasts or cells, with or withoutthe aid of electroporation, polyethylene glycol or other agents orprocesses known to alter membrane permeability to macromolecules.

The nucleic acid fragments of the invention can be used to transformprotoplasts or cell cultures from a wide range of higher plant speciesto form plant tissue cultures of the present invention. These speciesinclude the dicotyledonous plants tobacco, petunia, cotton, sugarbeet,potato, tomato, lettuce, melon, sunflower, soybean, canola (rapeseed)and other Brassica species and poplars: and the monocotyledonous plantscorn, wheat, rice, Lolium multiflorum and Asparagus officinalis. It isexpected that all protoplast-derived plant cell lines can be stablytransformed with the fragments of the invention.

The nucleic acid fragments of the invention can also be introduced intoplant cells with subsequent formation of transformed plants of thepresent invention. Transformation of whole plants is accomplished inplants whose cells can be transformed by foreign genes at a stage fromwhich whole plants can be regenerated. In the present invention,transformed plants are monocotyledonous and dicotyledonous plants.Preferably, the transformed plants are selected from the groupconsisting of tobacco, petunia, cotton, sugarbeets, potato, tomato,lettuce, sunflower, soybean, canola and other Brassica species, poplars,alfalfa, clover, sugarcane, barley, oats and millets: see "Handbook ofPlant Cell Culture" Vols. 1-3, Evans, D. A. et al., Sharp et al., andAmmirato et al., respectively, MacMillan, N.Y. (1983, 84). Mostpreferably, the transformed plants are selected from the groupconsisting of tobacco, petunia, potato, tomato, sunflower, sugarbeet,alfalfa, lettuce or Brassica species. The range of crop species in whichforeign genes can be introduced is expected to increase rapidly astissue culture and transformation methods improve and as selectablemarkers such as the fragments of the invention (see discussion below)become available.

One could further increase the level of expression of the nucleic acidfragments of the invention by replacing their native regulatorynucleotide sequences, 5' and 3' to the ALS coding sequence, withsynthetic or natural sequences known to provide high level and/or tissuespecific expression. One may also substitute the nucleotide sequences ofthe nucleic acid fragments of the invention with other synthetic ornatural sequences which encode transit peptides which will allowefficient chloroplast uptake of the nucleic acid fragments of theinvention.

The nucleic acid fragments of the present invention also have utility asselectable markers for both plant genetic studies and plant celltransformations. A gene of interest, generally conferring someagronomically useful trait, e.g. disease resistance, can be introducedinto a population of sensitive plant cells physically linked to anucleic acid fragment of the present invention. Cells can then be grownin a medium containing a herbicide to which the ALS encoded by afragment of the invention is resistant. The surviving (transformed)cells are presumed to have acquired not only the herbicide resistancephenotype, but also the phenotype conferred by the gene of interest. Thenucleic acid fragments can be introduced by cloning vehicles, such asphages and plasmids, plant viruses, and by direct nucleic acidintroduction. Subsequently, in a plant breeding program, theagronomically useful trait can be introduced into various cultivarsthrough standard genetic crosses, by following the easily assayedherbicide resistance phenotype associated with the linked selectablegenetic marker.

Transformed plants of the present invention are resistant to many of thesulfonylurea, triazolopyrimidine sulfonamide and imidazolinoneherbicides. These herbicides are disclosed in the following patents andpublished patent applications as follows:

Sulfonylureas

    ______________________________________                                        U.S. 4,127,405      U.S. 4,383,113                                            U.S. 4,169,719      U.S. 4,394,153                                            U.S. 4,190,432      U.S. 4,394,506                                            U.S. 4,214,890      U.S. 4,420,325                                            U.S. 4,225,337      U.S. 4,452,628                                            U.S. 4,231,784      U.S. 4,481,029                                            U.S. 4,257,802      U.S. 4,586,950                                            U.S. 4,310,346      U.S. 4,435,206                                            U.S. 4,544,401      U.S. 4,514,212                                            U.S. 4,435,206      U.S. 4,634,465                                                                EP-A-204,513                                              ______________________________________                                    

Triazolopyrimidine sulfonamides

South African Application 84/8844 (published May 14, 1985)

Imidazolinones

U.S. Pat. No. 4,188,487

EP-A-41,623 (published Dec. 16, 1981)

The nucleic acid fragments of the present invention encode ALS which isresistant to the following sulfonylurea herbicides: ##STR2## wherein Ris H or CH₃ ;

J is ##STR3## R₁ is Cl, Br, NO₂, C₁ -C₄ alkyl, C₂ -C₄ alkenyl, CF₃, C₁-C₄ alkoxy, C₁ -C₄ haloalkoxy, C₃ -C₄ alkenyloxy, C₂ -C₄ haloalkenyloxy,C₃ -C₄ alkynyloxy, CO₂ R₉, CONR₁₀ R₁₁, S(O)_(m) R₁₂, OSO₂ R₁₂, phenyl,SO₂ N(OCH₃)CH₃, SO₂ NR₁₀ R₁₁, ##STR4## R₂ is H, Cl, Br, F, CH₃, NO₂,SCH₃, OCF₂ H, OCH₂ CF₃ or OCH₃ ;

R₃ is Cl, NO₂, CO₂ CH₃, CO₂ C₂ H₅, SO₂ N(CH₃)₂, SO₂ CH₃ or SO₂ C₂ H₅ ;

R₄ is C₁ -C₃ alkyl, Cl, Br, NO₂, CO₂ R₉, CON(CH₃)₂, SO₂ N(CH₃)₂, SO₂N(OCH₃)CH₃ or S(O)_(m) R₁₂ ;

R₅ is C₁ -C₃ alkyl, C₄ -C₅ cycloalkylcarbonyl, F, Cl, Br, NO₂, CO₂ R₁₄,SO₂ N(CH₃)₂, SO₂ R₁₂ or phenyl;

R₆ is H, C₁ -C₃ alkyl, or CH₂ CH═CH₂ ;

R₇ is H, CH₃, OCH₃, Cl or Br;

R₈ is H, F, Cl, Br, CH₃, OCH₃, CF₃, SCH₃ or OCF₂ H;

R₉ is C₁ -C₄ alkyl, C₃ -C₄ alkenyl or CH₂ CH₂ C₁ ;

R₁₀ is H or C₁ -C₃ alkyl;

R₁₁ is H or C₁ -C₂ alkyl;

R₁₂ is C₁ -C₃ alkyl;

R₁₃ is H or CH₃ ;

R₁₄ is C₁ -C₃ alkyl or CH₂ CH═CH₂ ;

m is 0, 1 or 2;

n is 1 or 2;

Q is CH₂, CHCH₃ or NR₁₅ ;

R₁₅ is H or C₁ -C₄ alkyl;

P is O or CH₂ ;

R₁₆ is H or CH₃ ;

R₁₇ is C(O)NR₁₈ R₁₉ ;

R₁₈ is H or CH₃ ;

R₁₉ is CH₃ ;

R₂₀ is H, Cl, F, Br, CH₃, CF₃, OCH₃ or OCF₂ H;

R₂₁ is H or CH₃ ;

X is CH₃, OCH₃, OC₂ H₅ or NHCH₃ ;

Y is CH₃, C₂ H₅, OCH₃, OC₂ H₅, OCF₂ H, OCH₂ CF₃, Cl, CH₂ OCH₃ orcyclopropyl;

Z is CH or N;

and their agriculturally suitable salts; provided that

a) when Y is Cl, then Z is CH and X is OCH₃ ;

b) when Y is OCF₂ H, then Z is CH;

c) when J is J-1 and R₁ is OSO₂ R₁₂ or phenyl, then Y is other than OCF₂H;

d) when J is J-2, then Y is other than OCF₂ H or OCH₂ CF₃ ; and

e) when J is J-3 and R₄ is S(O)_(m) R₁₂, then Y is other than OCH₂ CF₃.

Sulfonylurea herbicides to which the ALS is particularly resistantinclude

1) Compounds of Formula I where

J is J-1;

R₁ is Cl, CH₃, C₁ -C₄ alkoxy, C₁ -C₂ haloalkoxy, allyloxy, propargyloxy,CO₂ R₉, CONR₁₀ R₁₁, SO₂ N(OCH₃)CH₃, SO₂ NR₁₀ R₁₁, S(O)_(m) R₁₂, OSO₂R₁₂, phenyl or ##STR5## 2) Compounds of Formula I where J is J-2;

R is H; and

R₃ is SO₂ N(CH₃)₂, CO₂ CH₃ or CO₂ C₂ H₅.

3) Compounds of Formula I where

J is J-3

R is H; and

R₄ is CO₂ CH₃ or CO₂ C₂ H₅ ;

4) Compounds of Formula I where

J is J-4;

R is H;

R₅ is Cl, Br, CO₂ CH₃, CO₂ C₂ H₅ or ##STR6## R₆ is CH₃ ; and R₇ is H, Clor OCH₃ ;

5) Compounds of Formula I where

J is J-5;

R is H;

R₅ is CO₂ CH₃ or CO₂ C₂ H₅ ; and

R₇ is H or CH₃.

6) Compounds of Formula I where

J is J-6;

Q is CHCH₃ or NR₁₅ ;

R is H; and

R₈ is H, F, Cl, CH₃, OCH₃, CF₃ or SCH₃.

7) Compounds of Formula I where

J is J-7;

R is H;

P is O; and

R₈ is H, F, Cl, CH₃, OCH₃, CF₃ or SCH₃.

8) Compounds of Formula I where

J is J-8;

R is H;

R₁₆ is CH₃ ; and

R₈ is H, F, Cl, CH₃, OCH₃, CF₃ or SCH₃.

9) Compounds of Formula I where

J is J-9;

R is H; and

R₁₇ is C(O)N(CH₃)₂.

10) Compounds of Formula I where

R is H;

R₁ is Cl, C₁ -C₄ alkoxy, OCF₂ H, OCH₂ CH₂ Cl, CO₂ R₉, CON(CH₃)₂, SO₂N(CH₃)₂, SO₂ R₁₂ or OSO₂ R₁₂ ; and

R₂ is H, Cl, CH₃, or OCH₃.

The nucleic acid fragments of the present invention encode ALS which isresistant to the following triazolopyrimidine sulfonamides: ##STR7##wherein Ar is ##STR8## R_(a) is C₁ -C₄ alkyl, F, Cl, Br, I, NO₂,S(O)_(p) R_(d), COOR_(e) or CF₃ ;

R_(b) is H, F, Cl, Br, I, C₁ -C₄ alkyl or COOR_(e) ;

R_(c) is H, C₁ -C₄ alkyl, F, Cl, Br, I, CH₂ OR_(d), phenyl, NO₂ orCOOR_(e) ;

R_(d) is C₁ -C₄ alkyl;

R_(e) is C₁ -C₄ alkyl, C₁ -C₄ alkenyl, C₁ -C₄ alkynyl, or 2-ethoxyethyl;

V is H, C₁ -C₃ alkyl, allyl, propargyl, benzyl or C₁ -C₃ alkylcarbonyl;

X₁, Y₁, and Z₁, are independently H, F, Cl, Br, I, C₁ -C₄ alkyl C₁ -C₂alkylthio or C₁ -C₄ alkoxy; and

p is 0, 1 or 2.

Triazolopyrimidinesulfonamide herbicides to which the ALS isparticularly resistant include

1) Compounds of Formula II where

V is H.

2) Compounds of Preferred 1 where

X₁ is H or CH₃ ;

Y₁ is H;

Z₁ is CH₃ ; and R_(a) and R_(c) are not simultaneously H.

The nucleic acid fragments of the present invention encode ALS which isresistant to the following imidazolinones: ##STR9## wherein A is##STR10## R_(f) is C₁ -C₄ alkyl; R_(g) is C₁ -C₄ alkyl or C₃ -C₆cycloalkyl;

A₁ is COOR_(i), CH₂ OH or CHO;

R_(i) is H; C₁ -C₁₂ alkyl optionally substituted by C₁ -C₃ alkyl, C₃ -C₆cycloalkyl or phenyl; C₃ -C₅ alkenyl optionally substituted by phenyl or1-2 C₁ -C₃ alkyl, F, Cl, Br or I; or C₃ -C₅ alkynyl optionallysubstituted by phenyl or 1-2 C₁ -C₃ alkyl, F, Cl, Br or I;

B is H; C(O)C₁ -C₆ alkyl or C(O)phenyl optionally substituted by Cl, NO₂or OCH₃ ;

X₂ is H, F, Cl, Br, I, OH or CH3;

Y₂ and Z₂ are independently H, C₁ -C₆ alkyl, C1-C₆ alkoxy, F, Cl, Br, I,phenyl, NO₂, CN, CF₃ or SO₂ CH₃ ;

X₃ is H, C₁ -C₃ alkyl, F, Cl, Br, I or NO₂ ; and

L, M, Q and R_(h) are independently H, F, Cl, Br, I, CH₃, OCH₃, NO₂,CF₃, CN, N(CH₃)₂, NH₂, SCH₃ or SO₂ CH₃ provided that only one of M or Qmay be a substituent other than H, F, Cl, Br, I, CH₃ or OCH₃.

Imidazolinone herbicides to which the ALS is particularly resistantinclude

1) Compounds of Formula III where

B is H; and

A₁ is COOR_(i).

2) Compounds of Preferred 1 where

R_(f) is CH₃ ;

R_(g) is CH(CH₃)₂ ;

X is H;

Y₂ is H, C₁ -C₃ alkyl or OCH₃ ;

Z₂ is H;

X₃ is H, CH₃, Cl or NO₂ ; and

L, M, Q and R_(h) are H.

Any of the aforementioned compounds may be applied alone or incombination to the site, pre- and/or post-emergence. Because the cropplant itself is resistant to the herbicide(s), the spectrum of herbicideactivity can be chosen for its efficacy in controlling the unwantedvegetation.

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius;unless otherwise stated. It should be understood that these examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above disclosure and these examplesone skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

EXAMPLE I

Tobacco (Nicotiana tabacum cv. Xanthi) DNA from the Hra mutant was madeaccording to the procedure of Dunsmuir et al. (J. Mol. App. Genetics,1983, 2, 285). 2×13 g of 1-2 inch tobacco leaves were removed fromplants and immediately ground in 2×20 mL buffer A (10 mM Tricine-KOH pH7.6-1.14M sucrose--5 mM MgCl₂ --5 mM 2-mercaptoethanol) in the coldroom, using mortars and pestles. An additional 40-50 mL of buffer A wasadded, and the slurries were filtered through 16 layers of cheesecloth.The filtrates were centrifuged at 2500 rpm in a Sorvall GSA rotor at 4°C. for 5 minutes. The pellets were resuspended in 10 mL buffer A,another 100 mL of buffer A was mixed in, and the cells were centrifugedas above. The pellets were then resuspended in 100 mL buffer A +0.4%Triton X-100, and left on ice for 10 minutes, and centrifuged as above.The pellets were washed twice more in the latter buffer. The finalpellets were resuspended in 5 mL of resuspension buffer (50 mM Tris HClpH 8, 20 mM EDTA), 1 mL of resuspension buffer--10% sarkosyl was added,and the volumes were then adjusted to 10 mL with resuspension buffer.Proteinase K was added to 100 μg/mL to the lysates, and the lysates weredigested at 37° C. overnight. The lysates were then brought to a densityof 1.55 g/mL CsCl, and to a final concentration of 300 μg/mL ethidiumbromide. The solutions were centrifuged in a Beckman Ti70.1 rotor at40000 rpm at 15° C. for 24 hours, and the fluorescent DNA band wasremoved after visualization with long-wave UV light. To remove the DNA,holes were punched in the sides of the polyallomer tubes with an 18gauge needle, and the viscous DNA was allowed to drip into collectiontubes. Great care was taken at all stages after cell lysis to preventshearing of the DNA. The DNA was again gently resuspended in a CsClsolution of 1.55 g/mL density and 300 μg/mL ethidium bromide, andcentrifuged at 40000 rpm at 15° C. for 48 hours, in a Sorvall TFT65.13rotor. The DNA was again collected by side puncture of the tube. It wasgently extracted 10 times with TE (10 mM Tris HCl pH 8, 1 mM EDTA)saturated-isoamyl alcohol, and then dialyzed extensively against TE.

The standard techniques of recombinant DNA and molecular cloning usedhere are described in R. W. Davis, D. Botstein and J. R. Roth, AdvancedBacterial Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1980) and T. Maniatis, E. F. Fritsch and Sambrook, MolecularCloning:A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1982).

A tobacco DNA library was constructed following the procedures ofManiatis et al (see above). Tobacco DNA was digested with therestriction enzyme Sau 3A to give a majority of fragments in the 20kilobase size range, as assayed by agarose gel electrophoresis. Thefragments were loaded onto 10-40% sucrose (in 1 M NaCl, 20 mM Tris pH 8,1 mM EDTA) gradients and size-fractionated by centrifugation in aBeckman SW 28 rotor at 26000 rpm at 17° C. for 16 hours. Fractions fromthe sucrose gradients were collected and analyzed by agarose gelelectrophoresis, and fractions containing fragments in the 20 kilobasesize range were dialyzed against TE and ethanol precipitated. They werethen ligated to BamH I cut phage lambda EMBL3 arms, at a 2:1 molarratio, and packaged into lambda phage heads, following the instructionssupplied by the manufacturer of the lambda arms and packaging reactions(Stratagene Cloning Systems, San Diego, Calif.).

A tobacco DNA library of 400000 phage was plated on the host strain E.coli LE 392 (Silhavy, T. J., Berman, M. L. and Enquist, L. W. (1984),"Experiments with Gene Fusions," Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.) at a density of 50000 phage per 150 mm Petri dish,on 10 plates. Duplicate nitrocellulose filter lifts of the phage plaqueswere made according to the procedure of Maniatis et al., and werehybridized with ³² P-labeled probes carrying either 5' or 3' ALS genefragments produced in a riboprobe labeling system. Riboprobes weresynthesized according to the procedures accompanying the riboprobe kitsold by Promega Biotech (Madison, Wisc.). Plaques that gave positivesignals on films from both sets of filters were picked and thepurification process was reiterated, until well-isolated hybridizingplaques were obtained.

Minipreps of the DNA from plaque purified phage were analyzed byrestriction enzyme digestions. Two classes of cloned tobacco DNAfragment inserts were distinguished as shown in FIG. 1. Phages 1, 2, 17and 18 contained inserts related to the previously isolated ALS genefrom the SURA locus, encoding herbicide sensitive ALS. Phage 3 containedan insert distinct from the above which was expected to contain theSURB-Hra gene encoding herbicide resistant ALS. EcoR I fragments thatencompassed the hybridizing regions of phage 3 were subcloned into M13phage vectors and subjected to DNA sequence analysis, usingoligonucleotides to extend the sequenced regions in overlappingsegments. A single-open reading frame of 1992 nucleotides was found, andwas identified as an ALS gene by comparison of the deduced amino-acidSequence with conserved regions of the amino acid sequences of ALSproteins from other species.

ALS genes isolated from the herbicide-resistant mutant tobacco. Hra,were introduced into sensitive tobacco cells via the "binary vector"system employing Agrobacterium tumefaciens. The ALS genes were firstintroduced into a binary vector in A. tumefaciens via plasmidconjugation, and the engineered A. tumefaciens were then used totransform plant cells with the foreign genes via co-cultivation.

A) Introduction of the Isolated Tobacco ALS Genes into A. tumefaciens:

i) Construction of Binary Vectors: The standard techniques ofrecombinant DNA and molecular cloning used here are described in R. W.Davis, D. Botstein and J. R. Roth, Advanced Bacterial Genetics, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1980) and T.Maniatis, E. F. Fritsch and Sambrook, Molecular Cloning:A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982).The purified 8.3 kilobase Spe I nucleic acid fragment of the invention,which was isolated from the Hra tobacco mutant and which contains acoding sequence for a herbicide-resistant form of an ALS gene, wasinserted into the Xba I site of the plasmid vector pMuc19 (J. D. G.Jones, P. Dunsmiur and J. Bedbrook, EMBO Journal 4:2411-2418 (1985)).(Although, Spe I and Xba I restriction enzymes recognize different DNAsequences, the products of these digestions carry the same 5'overhanging sequence). The orientation of the insert fragment in one ofthe resultant plasmids, pAGS148, was determined by restriction enzymeanalyses (FIG. 2).

The binary vector pAGS135 was used to move plasmid pAGS148 into A.tumefaciens. Plasmid pAGS135 is derived from plasmid pAGS112 (P. Van denElzen, K. Y. Lee, J. Townsend and J. Bedbrook, Plant Mol. Biol.,5:149-154 (1985)) by digestion of plasmid pAGS112 DNA with Xho Irestriction endonuclease, treatment with the Klenow fragment of E. coliDNA polymerase I, and self-ligation of the DNA which effects the removalof the Xho I site outside the T-DNA Eight border. Plasmid pAGS112 isderived from the wide-host range vector pLAFR (A. M. Friedman, S. R.Long, S. E. Brown, S. E. Buikema and F. M. Ausubel, Gene, 18:289-296(1982)) by the insertion into pLAFR of an EcoR I fragment in which theT-DNA borders flank a gene for expressing kanamycin resistance in plantsand a unique BamH I site for cloning (Van den Elzen et. al., Plant Mol.Biol., 5:149-154 (1985)). CsCl purified plasmids pAGS148 and pAGS135were digested with BamH I, and the resultant BamH I-cleaved plasmidswere-ligated. The ligation mixtures were packaged into lambda phageparticles in vitro and used to infect Escherichia coli strain HB101.Transformants were selected on ampicillin. The physical map of arecombinant plasmid, pAGS152, from one of the transformants wasdetermined by restriction analyses and is shown in FIG. 2.

ii) Conjugation of Plasmid pAGS152 from E. coli into A. tumefaciens:Plasmid pAGS152 was introduced into A. tumefaciens by conjugationessentially by the three-way mating method of Ruvkun, G. and Ausubel, F.M., Nature, 289:85-88 (1981). E. coli strain HB101 harboring plasmidpAGS152 and E. coli strain HB101 harboring the mobilizing vector pRK2013(ATCC 37159) (D. Figurski and D. R. Helinski, Proc. Natl. Acad. SciU.S.A., 76:1648-1652 (1979)) were mixed with A. tumefaciens strainLBA4404 harboring plasmid pAL4404 (A. Hoekema, P. R. Hirsch, P. J. J.Hooykaas and R. A. Schilperoort, Nature, 303:179-180 (1983)) and allowedto mate on solid LB medium (J. H. Miller, Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1972)) at 28° C. for 16 hours. Transconjugants were selected on platescontaining rifampicin at 100 mg/liter and tetracycline at 1 mg/liter. A.tumefaciens LBA4404:pAGS152 was restreaked on minimal A mediumcontaining tetracycline at 1 mg/liter.

Essentially, a similar method was used to obtain both A. tumefaciensLBA4404 containing plasmid pAGS112, the binary vector without any plantnucleic acid fragment insert, and A. tumefaciens LBA4404 containingplasmid pAGS145, the binary vector containing a nucleic acid fragmentfrom phage clone 1. The latter fragment was also isolated from Hramutant tobacco plants and carries a gene for a herbicide-sensitive formof ALS; this gene is not the wild type allele of the gene for theherbicide-resistant ALS in the nucleic acid fragment of the inventionbut the SURA gene from the second genetic locus.

B) Introduction of the Isolated ALS Genes Into Sensitive Tobacco byCo-cultivation of the Plant Cells with A. tumefaciens LBA4404 (pAGS145)and LBA4404 (pAGS152).

All manipulations of sterile media and plant materials were done inlaminar flow hoods, under suitable containment. Plant growth and plantcell cultures were carried out at 27° C. All protoplast manipulationswere carried out at room temperature unless otherwise mentioned.

Day 1 (afternoon):

Protoplast isolation medium was prepared by adding the following toK3/S(1) Medium: 0.1% (w/v) of MES buffer (Sigma Chemical Co.), 1% (w/v)of Cellulase (Onozuka or Cellulysin), and 0.1% of Macerase (Onozuka).After gentle stirring for approximately 30 minutes, the pH was broughtto 5.6 with KOH and the medium was filter-sterilized.

Sterile tobacco (Nicotiana tabacum var. Wisconsin 38) plants werecultured from 1 cm apical or auxiliary explants on OMS Medium in MagentaBoxes under a cycle of a 16 hour light period (6,000-8,000 lux) followedby an 8 hour dark period. When the plants were 5-7 weeks old, fullyexpanded leaves (3-6 leaves down from the apex) were removed, and twoleaves each were placed, top surface down, on 20 mL of protoplastisolation medium in a 100×25 mm petri dish. The leaves were thensubmerged and finely divided with a sharp surgical blade. The midrib washeld and the cuts were made outward from it towards the leaf margin atapproximately 2 mm intervals. The petri dishes were then sealed withparafilm and the macerated tissue incubated overnight (14-17 hours) indarkness at 27-29° C. with gentle gyrotory agitation (20-25 rpm).

Day 2 (morning):

A 75 mm filtering funnel, lined with four layers of cheesecloth, wasclamped to a ringstand. A glass tube (approximately 15 cm long and withan outer diameter of <5 mm) was attached to the funnel with latextubing. The funnel, cheesecloth, latex and glass tubing were wrapped inaluminum foil and sterilized in an autoclave as a unit.

The glass tubing was placed in a Babcock bottle and the cheesecloth waswetted with K3/S(1) Medium. The digested leaf tissue from two petridishes was carefully poured into the funnel. The cheesecloth was rinsedand expressed into the bottle. The loaded bottles were topped withK3/S(1) Medium, covered with foil and centrifuged at approximately 100×gfor 10 minutes. The floating protoplasts (1-2 mL) were collected with a1 mL serological pipette and placed in 20 mL of K3/S(1) Medium inanother Babcock bottle. After resuspending the protoplasts by gentlyswirling, the Babcock bottles were topped and centrifuged as before. Thefloating protoplasts (1-2 mL) were collected as described above andplaced in 30 mL of K3/G(1) Medium. The protoplasts were counted in ahemacytometer, and the volume was adjusted to give 1×10⁺⁵protoplasts/mL. 5 mL aliquots of the protoplasts were plated in petridishes [100×20 mm tissue-culture petri dishes (Corning): these disheswere used in all subsequent protoplast manipulations] and cultured indarkness.

Day 2 (afternoon):

A single colony of A. tumefaciens, containing the desired planttransformation vector, viz., pAGS112 (plasmid vector above), pAGS152(containing the nucleic acid fragment of the present invention) orpAGS145 (containing a nucleic acid encoding a sensitive form of ALS),growing on a Minimal A plate was inoculated into 5 mL of Minimal AMedium in an 18 mm test tube and cultured overnight on a roller drum at40-60 rpm at 27°-28° C.

Day 3 (morning):

The optical density of the A. tumefaciens cultures was measured at 550nm and adjusted to 0.15 with Minimal A Medium, and the bacteria wereallowed to continue growing as described above.

Day 3 (afternoon):

When the optical density (at 550 nm) of the A. tumefaciens cultures was0.6 (log phase culture), approximately 6 hours after dilution, thebacteria were added to plant cells at a multiplicity of approximately 50bacteria/plant cell (an optical density of 1.0 at 550 nm=1.4×10⁹bacteria). The bacteria and plant cell mixture was co-cultivated for 66hours at 24° C. in low light (approximately 500 lux). Non-transformedprotoplast controls were incubated similarly, but without agrobacteria.The following protocol is carried out for each co-cultivation(transformed cells with different agrobacteria, as well asnon-transformed cells).

Day 6 (morning):

Co-cultivation was terminated by adding 20 mL of a 1:1 mixture ofK3/G(2) Medium:C Medium supplemented with 500 mg/liter of cefotaxime (toselect against the agrobacteria) to 5 mL of the co-cultivation mixture.The co-cultivated cells were gently and thoroughly resuspended in thenew medium by mixing with a 5 or 10 mL serological pipette. The celldensity was 2×10⁴ protoplast equivalents/mL (protoplast equivalents=initial protoplasts, assuming 100% recovery and cell survival) and theosmoticum was 0.35 M. Three 5 mL aliquots of each culture were dispensedinto fresh petri dishes.

From this juncture until the cells were embedded in solid medium, thecells were cultured in low light (500-1500 lux) without motion and wereaseptically transferred to different media. At the indicated times,cells from one plate of each culture were transferred to non-selectivemedia, while cells from the other two plates of each culture weretransferred to selective media containing either 50 mg/liter ofkanamycin or 2 ng/mL chlorsulfuron in order to select for transformedplant cells. For these transfers, the contents of each plate werecollected with a 5 mL serological pipette, placed in separate 15 mLpolystyrene conical centrifuge tubes and centrifuged at approximately50×g for 5-10 minutes. The supernatant fluid was removed with a pipettewithout disrupting the loose pellet. Pellets of co-cultivated cells fromeach plate were then gently resuspended in the appropriate fresh medium.

Day 10:

The cells were transferred into 5 mL of C Medium supplemented with 500mg/liter cefotaxime in the case of non-selected plant cells or with 500mg/liter cefotaxime and either 50 mg/liter of kanamycin or 2 ng/mLchlorsulfuron in the case of selected cells. Each of these cultures wasreturned to the petri dishes from which they were taken; in this way notall cells needed to be pelleted to effect a medium exchange with minimalcell loss.

Day 13:

Non-selected cells were transferred to 20 mL of a 3:1 mixture of CMedium:MSP Medium supplemented with 500 mg/liter of cefotaxime, and a 5mL aliquot was dispensed into a fresh petri dish (at a density of 5×10protoplast equivalents/mL). The selected cells were resuspended in 5 mLof a 3:1 mixture of C Medium:MSP Medium supplemented with 500 mg/litercefotaxime and 50 mg/liter kanamycin or 2 ng/mL chlorsulfuron andreturned to the original plates for further culture.

Day 16-17:

The cells were transferred to 5 mL of a 1:1 mixture of C Medium and MSPMedium supplemented with 500 mg/liter cefotaxime alone (in the case ofnon-selected plant cells) or with 500 mg/liter cefotaxime and either 50mg/liter of kanamycin or 2 ng/mL chlorsulfuron (in the case of selectedcells) and cultured as before.

Day 20:

The non-selected cells were transferred to 25 mL of 1:1 mixture of CMedium:MSP Medium, and the mixture added to 25 mL of a 1:1 mixture of a2 X MSP Medium and 1% (w/v) type VII agarose solution (50° C.). Theresultant culture was mixed quickly with a 25 mL wide-mouth serologicalpipette and dispensed in 5 mL aliquots into fresh petri dishes. Thesuspended micro calluses in the agar solution were spread carefully andevenly across the plates with agitation by hand. The plates were coveredand left in the hood for one hour to solidify before they were wrappedin parafilm and removed to the culture chamber. The cell density wasabout 5×10 protoplasts equivalents/mL and the osmoticum was 0.15M. Theembedded cells were counted on a colony counter approximately 10 dayslater (Tables 1 and 2, below).

The selected cells were transferred to 20 mL of a 1:3 mixture of CMedium:MSP Medium containing 50 mg/liter of kanamycin or 2 ng/mLchlorsulfuron. Five mL aliquots of the resuspended cultures (5×10⁺³protoplast equivalents/mL) were dispensed into four fresh petri dishesper selected culture and cultured as before.

Day 23-24:

Each 5 mL culture of the selected cells was diluted with 7.5 mL of MSPMedium supplemented with 50 mg/liter of kanamycin or 2 ng/mLchlorsulfuron in order to achieve a cell density of 2×10⁺³ protoplastequivalents/mL. This density-adjusted culture was mixed with 12.5 mL ofa 1:1 mixture of 2X MSP Medium and 1.0% (w/v) type VII agarose solution(50° C.) supplemented with 50 mg/liter of kanamycin or 2 ng/mLchlorsulfuron. Five mL aliquots of the mixed cultures were quicklydispensed with a 25 mL wide-mouth serological pipette into fresh petridishes. The final plating density was 1×10³ protoplast equivalents/mL,and the osmoticum of the culture was 0.1M. The plates were solidified asdescribed above. The embedded cells were scored for growth on a colonycounter approximately 10 days later.

Day 25+:

Ten to twelve individual transformed calluses/colonies were picked andtransferred with a No. 11 scalpel to a petri dish containing MSR mediumwith or without the appropriate selective agent for plant regeneration.The calluses were cultured at 27° C., with a photo period of 16 hours oflight (5000-8000 lux) followed by 8 hours of darkness. Shoots appearedafter 2-3 weeks and continued to be produced for several months. Shootsof 2-3 mm length were excised with a sharp surgical blade andtransferred for rooting to OMS Medium in Magenta boxes.

After root formation (1 to 4 weeks), plants were transferred to soil forregeneration by the methods of R. S. Chaleff and M. F. Parsons, Proc.Natl. Acad. Sci. -U.S.A. 75:5104 (1978), and B. Tisserat in Plant CellCulture: A Practical Approach, Ed. Dixon, R. A., IRL Press, Oxford(1985).

Results of Co-cultivation:

The results of the co-cultivation experiments show that the nucleic acidfragment of the invention--but not the tobacco SURA gene for theherbicide-sensitive form of ALS--confers herbicide resistance whenintroduced into herbicide-sensitive tobacco cells (Tables 4 and 5,below). Since the nucleic acid fragment of the invention can conferherbicide resistance at a similar frequency when introduced in eitherorientation with respect to the vector, it is believed to contain theregulatory sequences both 5' and 3' to the coding sequence which arerequired for the expression of the herbicide-resistant ALS gene.

The level of herbicide resistance conferred by the nucleic acid fragmentof the invention was determined by plating one hundred colonies each ofpAGS152 transformed N. tabacum cells resistant to chlorsulfuron at 2 ppband non-co-cultured wild type N. tabacum cells on differentconcentrations of chlorsulfuron. The number of colonies actively growingon different concentrations of chlorsulfuron after one month was scored(Table 6, below). While wild type colonies are sensitive tochlorsulfuron at 2 ppb, colonies derived from co-cultivation with A.tumefaciens containing pAGS152 could tolerate up to 2000 ppb. This levelof resistance of the transformants is comparable to that of the Hraherbicide-resistant mutant tobacco from which the nucleic acid fragmentof the invention was isolated, and it is about ten fold higher than thatof 84 herbicide-resistant mutant tobacco (parent of Hra).

                  TABLE 4                                                         ______________________________________                                        Transfer of DNA from Phage Clone 1 to                                         Sensitive N. tabacum Cells                                                    Number of colony forming units derived from                                   10.sup.5 protoplast equivalents one month after                               co-cultivation                                                                                N.t..sup.1                                                                           N.t./p145.sup.2                                        ______________________________________                                        no selection      3.5 × 10.sup.4                                                                   3.6 × 10.sup.4                               Kanamycin 50 μg/mL                                                                           0        5.9 × 10.sup.2                               Chlorsulfuron 2 ng/mL                                                                           0        0                                                  ______________________________________                                         .sup.1 Noncocultured (control) plant cells.                                   .sup.2 Plant cells cocultured with A. tumefaciens harboring pAGS145,          kanamycin resistance vector containing the tobacco gene for                   herbicidesensitive ALS from phage clone 1.                               

                  TABLE 5                                                         ______________________________________                                        Transfer of DNA from Phage Clone 3 to                                         Sensitive N. tabacum Cells                                                    Number of colony forming units derived from                                   10.sup.5 protoplast equivalents one month after                               co-cultivation                                                                            N.t..sup.1                                                                            N.t./p112.sup.2                                                                         N.t./p152.sup.3                                 ______________________________________                                        no selection  2.0 × 10.sup.4                                                                    2.0 × 10.sup.4                                                                    1.6 × 10.sup.4                        Kanamycin 50 μg/mL                                                                       0         2.5 × 10.sup.3                                                                    6.2 × 10.sup.2                        Chlorsulfuron 2 ng/mL                                                                       0         0         6.5 × 10.sup.2                        ______________________________________                                         .sup.1 Noncocultured (control) plant cells.                                   .sup.2 Plant cells cocultured with A. tumefaciens harboring pAGS112,          kanamycin resistance vector.                                                  .sup.3 Plant cells cocultured with A. tumefaciens harboring pAGS152,          kanamycin resistance vector containing phage clone 3.                    

                  TABLE 6                                                         ______________________________________                                        Level of Chlorsulfuron Resistance in Cells                                    of N. tabacum cv. W38 Transformed with Mutant ALS Gene                        Number of Colonies Actively Growing                                           After One Month on Selective Media.sup.1                                                               N. tabacum/.sup.3                                    chlorsulfuron (ppb)                                                                           N. tabacum.sup.2                                                                       mutant ALS gene                                      ______________________________________                                          0            100       100                                                    20           0         100                                                    50           0         100                                                   200           0         100                                                   500           0         100                                                   2000          0          99                                                  20000          N.D..sup.4                                                                               6                                                   50000          N.D..sup.4                                                                               0                                                   ______________________________________                                         .sup.1 one hundred colonies plated at each chlorsulfuron level.               .sup.2 Colonies derived from noncocultured (control) plant cells.             .sup.3 Colonies derived from cocultivation with A. tumefaciens harboring      pAGS152 and initially selected for chlorsulfuron resistance at 2 ppb.         .sup.4 Not determined.                                                   

    __________________________________________________________________________    N. tabacum Culture Media                                                      __________________________________________________________________________    Ingredient  Stock    [Final]                                                                              Amount/liter                                      __________________________________________________________________________    K.sub.3 Medium                                                                K.sub.3 Major salts                                                                       10X             100  mL                                           CaCl, .2H.sub.2 O                                                                         100X            10   mL                                           Fe EDTA     100X            10   mL                                           B5 vitamins 100X            10   mL                                           MS minors I 1000X           1    mL                                           MS minors II                                                                              1000X           1    mL                                           glucose     --       0.4M   72.08                                                                              gm                                           or                   0.4M   136.8                                             sucrose                                                                       K.sub.3 /S (1) - sucrose, phytohormone regime 1 (elevated)                    K.sub.3 /G (1) - glucose, phytohormone regime 1 (elevated)                    K.sub.3 /G (2) - glucose, phytohormone regime 2 (reduced)                     1 - NAA 3.0 mg/liter   2 - NAA 0.1 mg/liter                                     BAP 1.0 mg/liter       BAP 0.1 mg/liter                                        bring pH to 5.7, filter sterilize, and                                        store at 5°.                                                        C-Medium                                                                      C-Media majors                                                                            10X             100  mL                                           Fe EDTA     100X            10   mL                                           B5 vitamins 100X            10   mL                                           MS minors I 1000X           1    mL                                           MS minors II                                                                              1000X           1    mL                                           Mannitol             0.2M   36.44                                                                              gm                                           Sucrose              0.1M   34.2 gm                                           Mes buffer           3.0 mM 590  mg                                           NAA         1 mg/ml  0.1 mg/liter                                                                         100  ul                                           BAP         1 mg/ml  0.1 mg/liter                                                                         100  ul                                                       bring pH to 5.7, filter sterilize,                                            and store at 5°.                                           MSP-Medium (for cell proliferation)                                           MS majors   10X             100  mL                                           Fe EDTA     100X            10   mL                                           B5 vitamins 100X            10   mL                                           MS minors I 1000X           1    mL                                           MS minors II                                                                              1000X           1    mL                                           Sucrose              0.1M   34.2 gm                                           Mes buffer           3.0 mM 590  mg                                           NAA         1 mg/ml  0.1 mg/liter                                                                         100  ul                                           BAP         1 mg/ml  0.1 mg/liter                                                                         100  ul                                                       bring pH to 5.7, filter sterilize,                                            and store at 5°.                                           MSR-Medium (for plant regeneration)                                           MS major    10X             100  mL                                           Fe EDTA     100X            10   mL                                           B5 vitamins 100X            10   mL                                           MS minors I 1000X           1    mL                                           MS minors II                                                                              1000X           1    mL                                           Sucrose              0.1M   34.2 gm                                           Mes buffer           3.0 mM 590  mg                                           NAA         1 mg/ml  0.1 mg/liter                                                                         100  ul                                           BAP         1 mg/ml  1.0 mg/liter                                                                         1.0  mL                                                       bring pH to 5.7, ---> add agar                                    Agar (T.C.)          0.8% (w/v)                                                                           8.0  gm                                           autoclave                                                                     Kanamycin   50 mg/ml 50 ug/ml                                                                             1.0  mL                                           Sulfate     1 mg/ml  as desired                                                                           --                                                or          in 5 mM                                                           Chlorsulfuron                                                                             KOH                                                               Dispense 25 mL/100 × 25 mm petri dish and, if required,                 aseptically add selective agents when media has cooled                        to 50°.                                                                OMS-Medium (for plant maintenance)                                            MS majors   10X             100  mL                                           Fe EDTA     100X            10   mL                                           MS minors I 1000X           1    mL                                           MS minors II                                                                              1000X           1    mL                                           B5 vitamins 100X            10   mL                                           Sucrose              3.0% w/v                                                                             30   gm                                           Mes buffer           3.0 mM 590  mg                                                       pH 5.7, ---> add agar                                             Agar (T.C.)          0.8% (w/v)                                                                           8.0  gm                                            autoclave, dispense 50 ml/3" × 4" Magenta Box                          Minimal A Medium                                                              K.sub.2 HPO.sub.4           10.5 g                                            KH.sub.2 PO.sub.4           4.5  g                                            (NH).sub.2 SO.sub.4         1.0  g                                            Sodium citrate 2H.sub.2 O   0.5  g                                                        autoclave in 990 mL                                               MgSO.sub.4 7H.sub.2 O                                                                     1M       1 mM   1.0  mL add                                                                        sterile                                      Glucose     20%             10.0 mL add                                                                        sterile                                      To solidify media: autoclave agar (15 gm/liter) Difco                         Bacto. in separate 500 mL volume. Then mix salts and                          agar before dispensing.                                                       __________________________________________________________________________    Stock       Ingredient                                                                             [Final]                                                                              Amount/Liter                                      __________________________________________________________________________    MS major salts (10X)                                                                      NH.sub.4 NO.sub.3                                                                      20.6 mM                                                                              16.5 g                                                        KNO.sub.3                                                                              18.8 mM                                                                              19.0 g                                                        MgSO.sub.4 7H.sub.2 O                                                                  1.5 mM 3.7  g                                                        KH.sub.2 PO.sub.4                                                                      1.25 mM                                                                              1.7  g                                                        CaCl.sub.2 2H.sub.2 O                                                                  3.0 mM 4.4  g                                            C-Medium major salts                                                                      NH.sub.4 NO.sub.3                                                                      5.0 mM 4.0  g                                            (10X)       KNO.sub.3                                                                              15.0 mM                                                                              15.2 g                                                        MgSO.sub.4 7H.sub.2 O                                                                  3.0 mM 7.4  g                                                        KH.sub.2 PO.sub.4                                                                      0.5 mM 0.68 g                                                        CaCl.sub.2 2H.sub.2 O                                                                  3.0 mM 4.4  g                                            K Medium major salts                                                                      KNO.sub.3       25.0 g                                            (10X)       (NH.sub.4).sub.2 SO.sub.4                                                                     1.34 g                                                        MgSO.sub.4 7H.sub.2 O                                                                         2.5  g                                                        KH.sub.2 PO.sub.4                                                                             2.01 g                                                        NH.sub.4 NO.sub.3                                                                             2.5  g                                            CaCl.sub.2 2H.sub.2 O (100X)                                                              CaCl.sub.2.2H.sub.2 O                                                                         92.3 g                                            Fe-EDTA (100X)                                                                            Na.sub.2 EDTA   3.73 g                                                        FESO.sub.4.7H.sub.2 O                                                                         2.78 g                                            (dissolve EDTA entirely before adding FeSO.sub.4 ; pH to 3.0)                 Ms minor I (1000X)                                                                        H.sub.3 BO.sub.3                                                                              0.620                                                                              g                                                        MnCl.sub.2.4H.sub.2 O                                                                         1.980                                                                              g                                                        ZnSO.sub.4.7H.sub.2 O                                                                         0.920                                                                              g                                            MS minor II (1000X)                                                                       KI              83   mg                                                       Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                 25   mg                                                       CuSO.sub.4.5H.sub.2 O                                                                         2.5  mg                                                       CoCl.sub.2.6H.sub.2 O                                                                         2.16 mg                                           B5 vitamins (100K)                                                                        nicotinic acid  0.1  g                                                        thiamin HCl     1.0  g                                                        pyridoxine HCl  0.1  g                                                        myo-inositol    10.0 g                                            NAA         Naphthelene     1.0  g                                                        acetic acid                                                                   1 mg/mL                                                                       (dissolve in dilute                                                           KOH)                                                              BAP         Benzylamino     1.0  g                                                        purine 1 mg/mL                                                                (dissolve in                                                                  dilute HCl)                                                       __________________________________________________________________________    Supplemental Apparatus, Chemicals & Media                                     MES buffer         Sigma No. M-8250                                           (2 [N-Morpholino]                                                             ethanesulfonic Acid)                                                          Agarose Type VII Low                                                                             Sigma No. A-4018                                           Gelling Temperature                                                           (stock maintained                                                             molten at 50°)                                                         Cellulysin ™    Calbiochem 219466                                          Macerase ™      Calbiochem 441201                                          Cefotaxime, sodium salt                                                                          Calbiochem 219380                                          dilute w/g.d., sterile H.sub.2 O                                              store @ 5°, dark. < 10 day                                             as 50 mg/mL stock                                                             Kanamycin Sulfate  Sigma No. K-4000                                           dilute w/g.d., H.sub.2 O, filter sterile                                      store @ -20°, dark as 50 gm/mL stock                                   Chlorsulfuron      E. I. du Pont                                                                 de Nemours and                                                                Company, Wilmington,                                                          Delaware 19898                                             100 mm × 20 mm tissue culture                                                              Corning 25020                                              petri dish                                                                    Babcock bottle     Kimble                                                     Centrifuge (Babcock compatible)                                                                  Damon/IEC                                                                     Division HN-SII                                            Magenta Boxes 3" × 4"                                                                      Magenta Corp.                                                                 4149 W. Montrose Ave                                                          Chicago. IL 68641                                          T.C. Agar          KC Biological CR-100                                       __________________________________________________________________________

EXAMPLE II

Tobacco DNA from the C3 mutant was prepared and a genomic DNA library inbacteriophage vector EMBL3 was constructed as described in EXAMPLE I.Phage carrying ALS genes were identified, as described in EXAMPLE I, byhybridization to a ³² P-labeled 5' tobacco ALS gene fragment probe.

Six independent recombinant phage were isolated in a screen of 600,000recombinants from the C3 library. Restriction endonuclease analysis ofthese isolated phage indicated that the DNA inserts of three phage couldbe aligned with the SURA gene from the Hra library (phages 35, 36 and38). The remaining three phage (phage 31, 34 and 37) had DNA insertscorresponding to the SURB gene. It was expected that the ALS genecarried on phages 35, 36 and 38 would be the SURA-C3 gene, encodingherbicide resistant ALS and the ALS gene carried on phages 31, 34 and 37would be the SURB gene, encoding herbicide sensitive ALS.

DNA fragments from phages 31, 35 and 38 were subcloned into the pUC119plasmid and subsequently into the pAGS135 binary vector essentially asdescribed in EXAMPLE I. An approximately 8.3 kb Spe I restrictionendonuclease fragment from phage 31, analogous to that present inpAGS148 (FIG. 2), but carrying the SURB gene encoding herbicidesensitive ALS, was subcloned in both possible orientations in thevector. An approximately 6.3 kb Spe I-Sal I restriction endonucleasefragment from phage 35 and an approximately 7.8 kb Spe I-Sal I fragmentfrom phage 38 were subcloned yielding pALS35(ATCC #67424) and pALS38,respectively. The fragments included 2.5 kb in the 5' direction(upstream) of the ALS coding region, 2.0 kb of ALS coding sequence,encoding herbicide resistant enzyme and 1.8 and 3.3 kb, respectively inthe 3' direction (downstream) from the ALS coding region. The latter twosubcloned fragments contain a BamH I restriction endonuclease site.Partial BamH I digestions or pALS35 and pALS38 were employed forinsertion of these plasmids into the BamH I site of the binary vectorpAGS135. The ALS genes in the binary vector, designated p312, p313, p351and p381 (Table 7) were moved into A. tumefaciens by tri-parentalmating, as described in EXAMPLE I.

Introduction of the ALS genes into herbicide sensitive tobacco byco-cultivation of plant cells with A. tumefaciens carrying the ALS genesin the binary vector was performed as described in EXAMPLE I. Theresults of these co-cultivation experiments are shown in Table 7. TheALS gene isolated in phage 31, i.e. the SURB gene encoding herbicidesensitive ALS, yielded no herbicide resistant plant cells, as expected.The ALS gene isolated in phages 35 and 38, i.e. the SURA-C3 geneencoding herbicide resistant ALS did yield herbicide resistant plantcells. Herbicide resistant plant cells arose at a lower frequency thankanamycin resistant plant cells and at a lower frequency than wasobserved when the SURB-Hra gene was used. This may reflect either thelesser resistance to the herbicide of the ALS enzyme encoded by theSURA-C3 gene compared to that encoded by the SURB-Hra gene, or the lowerexpression of the SURA-C3 gene compared to the SURB-Hra gene, or both.

                                      TABLE 7                                     __________________________________________________________________________    ALS Reintroduction Exp. 3                                                     Transfer of DNA from phage clones 31, 35 & 36                                 to Sensitive N. tabacum Cells                                                 Number of Colony Forming Units derived from 10.sup.5                          Protoplast Equivalents One Month after Co-cultivation                                         N.t./                                                                              N.t./                                                                              N.t./                                                                              N.t./                                                                              N.t./                                                N.t..sup.1                                                                         p152.sup.2                                                                         p312.sup.3                                                                         p313.sup.4                                                                         p351.sup.5                                                                         p381.sup.6                                __________________________________________________________________________    no selection                                                                             2.1 × 10.sup.4                                                               1.5 × 10.sup.4                                                               1.1 × 10.sup.4                                                               1.9 × 10.sup.4                                                               1.5 × 10.sup.4                                                               1.4 × 10.sup.4                      Kanamycin 50 ug/ml                                                                       0    88   65   48   139  87                                        Chlorsulfuron 2 ng/ml                                                                    0    83    0    0    32  18                                        __________________________________________________________________________     .sup.1 Non cocultured plant cells.                                            .sup.2 Plant cells cocultured with A. tumefaciens harboring pAGS152,          S4/Hra subclone.                                                              .sup.3 Plant cells cocultured with A. tumefaciens harboring pAGS312, C3       subclone, φ31 (orientation 1).                                            .sup.4 Plant cells cocultured with A. tumefaciens harboring pAGS313, C3       subclone, φ31 (orientation 2).                                            .sup.5 Plant cells cocultured with A. tumefaciens harboring pAGS351, C3       subclone, φ35.                                                            .sup.6 Plant cells cocultured with A. tumefaciens harboring pAGS381, C3       subclone, φ38.                                                       

EXAMPLE III

Mutations were made in the wild-type SURA gene of tobacco in vitro inorder to make it encode a herbicide resistant ALS. Restrictionendonuclease fragments containing part of the SURA gene were subclonedinto M13 phage vectors or plasmid vectors containing an M13 origin ofreplication to allow production of single-stranded DNA. The specific DNAfragment subcloned depended upon the region to be mutagenized in vitroand the availability of restriction endonuclease sites.

Oligonucleotides 16-17 bases in length, which hybridized to thesingle-stranded DNA from the SURA ALS gene with single base mismatches,were synthesized. These mismatches were designed to convert amino acidcodons found in the wild-type ALS gene to the codons found in ALS geneswhich encode ALS enzymes resistant to sulfonylurea herbicides. Theseoligonucleotides include 5' GTTCAATTGGAGGATC 3', to change trp 591 toleu, 5' GTCAAGTGGCACGTAGG 3', to change pro 197 to ala, and 5'GTCAAGTGTCACGTAGG 3', to change pro 197 to set, 5' ATGTACCTGAGGATATT 3'to change lys 256 to glu, 5' GAGGTTTGTTGATAGAG 3' to change asp 384 toval, 5' AGGTTTGAGGATAGAGT 3' to change asp 384 to glu, 5'TACTGATGATTTTCAGG 3' to change ala 205 to asp and 5' CAGGTGGCCCTTCCATG3' to change ala 122 to pro.

The oligonucleotides were hybridized to single-stranded DNA and used asprimers for synthesis of a complementary strand in a reaction catalyzedby the Klenow fragment of DNA polymerase I, following the procedures ofCarter et al. (Oligonucleotide site-directed mutagenesis in M13, AnglianBiotechnology Limited, England, 1985). The resulting DNA was transformedinto competent E. coli mutL cells (Kramer et al., 1984, Cell 38, 879).Individual plaques or colonies were purified, depending on whether M13phage vectors (M13mp18/19) or M13 replication origin plasmid vectors(pTZ18R/19R, Pharmacia; Piscataway, N.J.) were used. Mini-preps ofsingle-stranded DNA were made and used in DNA sequencing reactions toidentify clones that carried the mutated bases.

These in vitro constructed site-specific mutations can be incorporatedsingly or in combination into either a wild type SURA or SURB gene whichincludes the 5' and 3' regulatory sequences needed to provide expressionof the gene in plant cells (see EXAMPLES I and II). This is accomplishedby substituting restriction endonuclease fragments carrying themutations into a plasmid carrying the SURA or SURB gene from which theanalogous fragment has been removed. The choice of the restrictionfragment to substitute depends upon the position of the mutation in thegene and the availability of restriction endonuclease sites. Theintroduction of the mutated genes into plant cells can then beaccomplished as described in EXAMPLES I and II. Any of the DNA fragmentscontaining mutations which result in production of herbicide resistantALS, as disclosed in the description of the invention, can be producedessentially by this method. Furthermore, the mutations need not be madeexclusively in the SURA gene. Analogous mutations can be made in theSURB gene or any other plant gene encoding ALS for which DNA sequenceinformation is available.

Several different 1.4 kb Nco I to Bgl II DNA fragments from the in vitromutated SURA gene (nucleotide positions 533-1952 as indicated in FIG. 5)were inserted into the SURB gene (nucleotide positions 1234-2653 asindicated in FIG. 4) replacing the wild type SURB gene sequence.

The ability of these chimeric ALS genes to confer herbicide resistanceon plant cells was assayed by co-transforming tobacco protoplasts withplasmids carrying the mutated ALS genes and with a second plasmidcarrying a NOS-NPTII-NOS gene. Half of each transformation mixture wassubjected to selection on kanamycin and half to selection onchlorsulfuron. The recovery of chlorsulfuron-resistant colonies is shownas a percentage of kanamycin-resistant colonies in Table 8.

Protoplasts were prepared from Nicotiana tabacum cv. Xanthi by themethod of Nagy and Maliga [Callus induction and plant regeneration frommesophyll protoplasts of N. sylvestris. Z. Pflanzenphysiologie78:453-455 (1976)], as modified by Potrykus and Shillito [Protoplasts:isolation, culture, plant regeneration. In: Weissbach, A., Weissbach, H.(eds.) Methods in Enzymology 118: 549-578. Academic Press. (1985)]. DNAwas introduced into protoplasts as follows, after the methods of Krens[In vitro transformation of plant protoplasts with Ti-plasmid DNA.Nature 296:72-75 (1982)] and Shillito, [Agarose plating and a bead typeculture technique enable and stimulate development of protoplast derivedcolonies in a number of plant species. Plant Cell Reports 2: 244-247(1983); High efficiency direct gene transfer to plants. Biotechnology3:1099-1103 (1985)].

Protoplasts were resuspended at 1.5 million cells/ml in sterile 0.4Mmannitol, 6 mM MgCl2, 0.1% MES pH 5.8, and divided into aliquots of onemillion cells in sterile 50 ml centrifuge tubes. The cells weresubjected to a 45° C. heat shock for five minutes, and then quicklycooled on ice to room temperature.

Ten μg of the plasmid pKNKS carrying the NOS-NPTII-NOS gene, 10 μg ofthe appropriate ALS gene-carrying plasmid, and 30 μg of sheared calfthymus DNA were ethanol-precipitated, resuspended in 50 μl sterilewater, and then added to each aliquot of protoplasts. 40% PEG in 0.4 Mmannitol, 30 mM MgCl2, 0.1% MES pH 5.8 was added dropwise to each tube,by forcing the solution through a syringe-mounted 0.45 μm filter, to afinal concentration of 13% PEG. Tubes were swirled gently several timesduring a ten minute period to keep the phases mixed. Three 0.33 mlaliquots from each transformation mix were gently pipetted into 60×15 mmsterile culture dishes containing 3 ml sterile H Medium by the method ofPotrykus and Shillito (2) and swirled gently to mix. Dishes were sealedwith Parafilm and incubated three days in the dark and four days in thelight at 25° C.

Cells were resuspended by scraping the bottom of each dish with asterile cell scraper, and then 3.3 ml of 1.2% DNA-grade agarose in K3AMedium, autoclaved and cooled to 45° C., was added to each dish. Disheswere swirled immediately to mix phases. When the agarose had solidified,each disk was cut into quarters, thus producing twelve quarters fromeach transformation. Agarose quarters were floated, three per dish, in100×25 mm sterile culture dishes containing 25 ml of 1:1 K3A:H Mediumand the appropriate selective agent. Six quarters, representing 500,000cells in the original mix, from each transformation were floated in 50mg/L kanamycin and six were floated in 2 ppb chlorsulfuron. Dishes wereincubated in the light at 25° C.

After one week, one half of the liquid medium in each dish was replacedwith 1:1 K3A:H containing the appropriate selective agent. After onemore week, one half of the liquid medium was replaced with 1:1 K3E:Jcontaining the appropriate selective agent. Media were then replenishedbi-weekly with 1:1 K3E:J until colonies became visible. Final colonycount was at 6 weeks.

The data shown in Table 8 indicates that all of the in vitro mutated ALSgenes can be used to transform tobacco cells to herbicide resistance.However, the frequency of obtaining herbicide resistance varies widely;the reason for this is not yet known.

                                      TABLE 8                                     __________________________________________________________________________    Herbicide Resistant Transformants with                                        In Vitro Mutated ALS Genes                                                                      Colonies/1 Million                                                            Treated Cells                                                                 50 mg/                                                                             2 ppb    Cs-R/                                         ALS Gene          L Kan                                                                              Chloroulfuron                                                                          Kan-R (%)                                     __________________________________________________________________________    None              323  1        0.3                                           SURB-Hra.sup.1 : (Ala 197 + Leu 591)                                                            430  126      29.3                                          SURB/SURA.sup.2 : (wild type)                                                                   229  0        0.0                                           SURB/SURA: (Ala 197)                                                                            68   8        11.8                                          SURB/SURA: (Ala 197 + Leu 591)                                                                  78   39       50.0                                          SURB/SURA: (Ser 197)                                                                            205  80       39.0                                          SURB/SURA: (Ser 197 + Leu 591)                                                                  180  49       27.2                                          SURB/SURA: (Leu 591)                                                                            127  7        5.5                                           SURB/SURA: (Asp 205)                                                                            105  31       29.5                                          SURB/SURA: (Glu 256)                                                                            86   3        3.5                                           SURB/SURA: (Val 384)                                                                            80   1        1.2                                           __________________________________________________________________________     .sup.1 Gene isolated from Hra plants, carrying in vivo derived mutations.     .sup.2 Chimeric SURBSURA gene in which the NcoI to BglII fragment of SURB     is substituted with the equivalent fragment from SURA; no in vitro            mutations.                                                               

EXAMPLE IV

DNA was prepared from Beta vulgaris cv. Sennika (sugarbeet) and agenomic DNA library in bacteriophage lambda vector EMBL3 was constructedas described in EXAMPLE I. Recombinant phage (300,000) were screened byhybridization to a ³² P-labeled 5' ALS tobacco gene fragment probe asdescribed in EXAMPLE I. The filters were washed at 42° C. (0.1×SSC) and20 individual clones were isolated. On the second round of purificationthe recombinant phage were hybridized to both the 3' tobacco ALS geneprobe and the 5' probe. In addition, the filters which had beenhybridized to the 5' probe were washed at 55° C. Only one clone, φ21,hybridized to both 5' and 3' probes and also remained hybridized afterthe 55° C. wash. Minilysate DNA preparations were made from the 20clones and digested with EcoR I and Nco I. The different isolates haddifferent restriction endonuclease digestion patterns and again only φ21hybridized to both probes and remained hybridized after a 55° C. wash.One phage, φ41, also had a hybridizing band remaining after a 55° C.wash but it did not hybridize to the 3' probe. FIG. 7 shows therestriction endonuclease map of the phage φ21, together with subcloneswhich have been constructed from it. The ALS coding region was localizedto a 3.0 kb BamH I--Hind III fragment by hybridization with 5' and 3'probes from the N. tabacum gene. Both DNA strands of this fragment havebeen sequenced. The BamH I--Hind III fragment was subcloned into pUC119or Bluescript (Strategene; San Diego, Calif.) vectors; then ExonucleaseIII or Bal 31 deletions were generated. The dideoxy sequencing methodwas used. A comparison of the deduced amino acid sequence encoded by thesugarbeet gene with that of the tobacco gene(s) indicates no homology inthe first 88 amino acids of the predicted protein (see FIG. 8). Thisregion may represent the chloroplast transit peptide. Thereafter thehomology is approximately 90% with an insertion of 4 amino acids aroundresidue 290 of tobacco ALS. Inspection of the amino acid residues whichdefine the sites for herbicide resistance identified in tobacco andyeast indicate that these residues are conserved in sugarbeet ALS also.These data allow a straightforward approach to the construction of agene encoding herbicide resistant sugarbeet ALS enzyme, by site-directedmutagenesis, as described in EXAMPLE III.

Three sites have been mutagenized in this sugarbeet gene. The codon GCAfor ala at position 122 (numbering of amino acid residues from FIG. 6)was changed to CCA for pro, the codon CCA for pro at position 197 waschanged to GCA for ala and the codon TGG for trp at position 591 waschanged to TTG for leu. The double mutation yielding pro to ala at 197and trp to leu at 591, which mimics the tobacco SURB-Hra gene, was alsomade by combining the two single mutations.

In order to transform plants with these in vitro constructed mutationsin the sugarbeet ALS gene, DNA fragments containing the mutations andextending from the BamH I site about 910 bp in the 5' direction(upstream) of the coding region to the Pst I site about 1000 bp in the3' direction (downstream) of the coding region (see FIG. 17) were clonedinto plasmid vector pUC119. These were introduced into the binary vectorpAGS140 for transformation into plant cells as described in EXAMPLE I.The binary vector pAGS140 is similar to pAGS135 (FIG. 2) except thatbetween the BamH I site and the right border of the T-DNA of theTi-plasmid of Agrobacterium tumefaciens a gene which will conferresistance in plants to the antibiotic hygromycin was inserted.

Introduction of the ALS genes into herbicide sensitive tobacco andsugarbeet, by co-cultivation of the plant cells with A. tumefacienscarrying the ALS genes in the binary vector, was performed as describedin EXAMPLE I and EXAMPLE II. Results of a co-cultivation experiment intobacco are shown in Table 9. Herbicide resistant transformants wereobtained with three of the four mutant sugarbeet ALS genes. Thefrequency of obtaining herbicide resistant transformants was lower thanthat for kanamycin resistant transformants, and also lower than thefrequency of herbicide resistant transformants obtained when the tobaccoSURB-Hra gene was used. It is believed that this results from poorexpression of the mutant sugarbeet ALS genes in tobacco. This mayreflect either insufficient nucleotide regulatory sequences upstream ordownstream of the mutant sugarbeet ALS genes in the DNA fragments usedor poor utilization of sugarbeet nucleotide regulatory sequences intobacco, or both. The mutant sugarbeet ALS gene carrying the Ala 122 toPro substitution did not yield chlorsulfuron-resistant transformants asexpected, since it is a gene known to confer resistance to only selectedsulfonylurea herbicides which do not include chlorsulfuron.

                                      TABLE 9                                     __________________________________________________________________________    Herbicide Resistance in Plant Cells Transformed                               with Site Specific Mutant Sultarbeet ALS Genes                                Gene                                                                          Origin Tobacco                                                                              Sugarbeet                                                                            Sugarbeet                                                                           Sugarbeet                                                                            Sugarbeet                                   __________________________________________________________________________    Mutation                                                                             pro(197)-ala/                                                                        pro(197)-ala/                                                                        trp(591)-leu                                                                        pro(197)-ala                                                                         ala(122)-pro                                       trp(591)-leu                                                                         trp(591)-leu                                                    Chlor- 1500   254     24   106     0                                          sulfuron.sup.R                                                                Kanamycin.sup.R                                                                      2379   2682   2707  2376   892                                         Chl.sup.R /Kan.sup.R                                                                 .631   .095   8.9 × 10.sup.-3                                                               .045   --                                          __________________________________________________________________________     Gene introductions were done by standard cocultivation method. For each       construction an aliquot of the cocultured plant cells (2 × 10.sup.5     starting plant calls) wag scored for chlorsulfuron and another aliquot fo     kanamycin resistance.. Selection was with chlorsulforon at 2 ppb or           kanamycin at 50 ppm.                                                     

EXAMPLE V

The SURB-Hra gene described in EXAMPLE I was transformed into tobaccocultivars by Agrobacterium tumefaciens infection of tobacco leaf disksand progeny of the transformants were analyzed to demonstrate expressionof resistance at the whole plant level and inheritance of the herbicideresistance trait. Standard aseptic techniques for the manipulation ofsterile media and axenic plant/bacterial cultures were followed,including the use of a laminar flow hood for all transfers. Pottedtobacco plants for leaf disk infections were grown in a growth chambermaintained for a 12 hr, 24° C. day, 12 hr, 20° C. night cycle, withapproximately 80% RH, under mixed cool white fluorescent andincandescent lights. Tobacco leaf disk infections were carried outessentially by the method of Horsch, R. B., Fry, J. E., Hoffmann, N. L.,Eichholtz, D., Rogers, S. G., Fraley, R. T., (1985, Science 227:1229-1231.

Young leaves, not fully expanded and approximately 4-6 inches in length,were harvested with a scalpel from approximately 4-6 week old tobaccoplants (Nicotiana tabacum cv NK326 or K14). The leaves were surfacesterilized for 30 minutes by submerging them in approximately 500 ml ofa 10% Chlorox, 0.1% SDS solution and then rinsed 3 times with steriledeionized water. Leaf disks, 6 mm in diameter, were prepared from wholeleaves using a sterile paper punch.

Leaf disks were inoculated by submerging them for several minutes in 20ml of a 1:10 dilution of an overnight Agrobacterium culture carrying theplasmid pAGS152. Agrobacterium cultures were started by inoculating 10ml of Min A (EXAMPLE I) broth with a single bacterial colony removedfrom a Min A plus tetracycline (EXAMPLE VI) plate. The culture was grownfor approximately 17-20 hours in 18 mm glass culture tubes in a NewBrunswick platform shaker maintained at 28° C.

After inoculation, the leaf disks were placed in petri dishes containingCN agar medium (EXAMPLE VI). The dishes were sealed with parafilm andincubated under mixed fluorescent and "Gro and Sho" plant lights(General Electric) for 2-3 days in a culture room maintained atapproximately 25° C.

To rid the leaf disks of Agrobacterium and to select for the growth oftransformed tobacco cells, the leaf disks were transferred to fresh CNmedium containing 500 mg/1 cefotaxime and 100 mg/1 kanamycin. Cefotaximewas kept as a frozen 100 mg/ml stock solution and added aseptically(filter sterilized through a 0.45 μm filter) to the media afterautoclaving. A fresh kanamycin stock (50 mg/ml) was made for each useand was filter sterilized into the autoclaved media.

Leaf disks were incubated under the growth conditions described abovefor 3 weeks and then transferred to fresh media of the same composition.

Approximately 1-2 weeks later shoots developing on kanamycin-selectedexplants were excised with a sterile scalpel and planted in A mediumcontaining 100 mg/1 kanamycin. Root formation on selective andnon-selective media was recorded within 3 weeks. Shoots which rooted inkanamycin were transferred to soil and grown in a growth chamber asdescribed above. After 3 to 5 weeks, but before flowering had occurred,leaf tissue was excised and used for ALS assays as described in EXAMPLEVI. The results of these assays, which indicate that a herbicideresistant form of ALS was being produced, are shown in Table 10. Theplants exhibiting herbicide resistant ALS activity were then moved to agreenhouse where they were grown to maturity. Individual flowers werebagged to permit self-fertilization without cross-pollination. Matureseeds were harvested and progeny tests were conducted to determineinheritance of the herbicide resistance trait. Seeds were surfacesterilized as described above, dried and planted on SG medium (1/4 MSsalts, 0.75% sucrose, 0.8% Agar) in the presence or absence of herbicide(DPX-F6025, Classic®). Sensitive seeds germinated, but did not developfurther. Results of the progeny analyses are shown in Table 10. Asegregation ratio of 3 resistant progeny to 1 sensitive indicated thepresence of a single-site insertion of the SURB-Hra gene in the genomeof the transformant, which was stably inherited. This was seen in 15 of17 transformants. Higher ratios of resistant to sensitive progenyindicated multiple insertions at unlinked positions in the genome. The15/1 ratio indicates the presence of 2 unlinked SURB-Hra genes and the255/1 ratio indicates 4 unlinked SURB-Hra genes in the transformants K14#40 and K14 #7, respectively.

                                      TABLE 10                                    __________________________________________________________________________                    Progeny                                                              % Unhibited                                                                            Resistant/Sensitive.sup.2                                                                 Segregation Ratio                                        ALS Activity.sup.1                                                                     100 ppb                                                                             1000 ppb                                                                            Resistant/Sensitive                               __________________________________________________________________________    NK326(wt)                                                                             7       --    --    --                                                NK326 #1                                                                             36        98/37                                                                              90/35 3/1                                               NK326 #9c                                                                            47       163/49                                                                              99/63 3/1                                               NK326 #9d                                                                            37       288/67                                                                              150/58                                                                              3/1                                               NK326 #10                                                                            26        93/31                                                                              96/24 3/1                                               NK326 #10c                                                                           56       333/45                                                                              290/76                                                                              3/1                                               K14 wt  7       --    --    --                                                K14 #7 71       990/4 109/1 255/1                                             K14 #11                                                                              52       208/85                                                                              127/76                                                                              3/1                                               K14 #27                                                                              45       129/45                                                                              108/42                                                                              3/1                                               K14 #29                                                                              30       192/46                                                                              163/67                                                                              3/1                                               K24 #31                                                                              44       106/35                                                                              99/34 3/1                                               K14 #32c                                                                             32       140/65                                                                              63/86 3/1                                               K14 #40                                                                              41       218/18                                                                              212/26                                                                              15/1                                              K14 #41                                                                              40       255/35                                                                              296/74                                                                              3/1                                               K14 #42                                                                              29       162/74                                                                              77/72 3/1                                               K14 #53                                                                              37       130/59                                                                              149/139                                                                             3/1                                               K14 #54                                                                              34        99/38                                                                              92/43 3/1                                               K14 #54A                                                                             28       137/55                                                                              100/72                                                                              3/1                                               __________________________________________________________________________     .sup.1 The ALS activity in each line is related to the activity in the        absence of herbicide which is taken as 100 percent. The sulfonylurea          herbicide used was DPXF6025 (Classic ® ) at a concentration of 10 ppb     .sup.2 Resistant progeny are able to growth at the indicated                  concentrations of herbicide DPXF6025 (Classic ® ).                   

EXAMPLE VI

To transform herbicide sensitive tomato to resistance the SURB-Hra genefrom tobacco, carried on the binary vector pAGS152 in A. tumefaciensstrain LBA4404, was used (see EXAMPLE I).

Standard aseptic techniques for the manipulation of sterile media andaxenic plant/bacterial cultures were followed, including the use of alaminar flow hood for all transfers.

Seeds of tomato (Lycopersicon esculentum vat. Herbst Red Cherry) weresurface sterilized for 30 minutes in a 10% Chlorox, 0.1% SDS solutionand rinsed 3 times with sterile deionized water. The seeds were plantedin Magenta boxes (Magenta Corp.) containing 100 ml of OMS agar mediumand germinated under mixed fluorescent and "Gro and Sho" plant lights(General Electric) in a culture room maintained at approximately 25° C.Cotyledons from 10-15 day old seedlings were used for Agrobacteriuminoculation.

Cotyledons were wounded by removing approximately 2 mm of tissue fromeach end of the cotyledon with a sterile scalpel. Wounded cotyledonswere planted in petri dishes on CTM agar medium either with or without75μM acetosyringone (Aldrich Chemical).

In preparation for the cotyledon inoculation, a single bacterial colonyfrom a Min A+tetracycline (1 μg/ml) agar plate was inoculated into aflask containing 30 ml of Min A broth (EXAMPLE I) and grown for 2 daysat 28° C. in a New Brunswick platform shaker. On the morning of thecotyledon inoculation, the bacterial culture was diluted with sterileMin A broth to an OD of 0.1 at 650 nM and allowed to multiply to an ODof 0.2 under the growth conditions previously described. This culturewas then used undiluted for the inoculation.

CTM agar plates containing the colyledon explants were flooded with 5 mlof the bacterial solution for approximately 5 minutes, before removal Ofthe solution. The plates were then secured with Time Tape (ShamrockScientific Specialty) on two sides of the dish and incubated for 2 daysunder mixed fluorescent and "Gro and Sho" plant lights (GeneralElectric) at approximately 25° C. for two days.

To rid the plant cultures of Agrobacterium and to select for the growthof transformed tomato cells, the cotyledon explants were transferred tofresh CTM medium containing 500 mg/L cefotaxime and 50 mg/L kanamycinand incubated under the same culture conditions described above forapproximately 3 weeks. The cotyledons were than transferred to freshmedia of the same composition and selective agents as CTM but with 1/10the zeatin concentration.

After approximately 2-4 weeks, shoots developing off ofkanamycin-selected cotyledons were excised and planted in OMS mediacontaining 500 mg/L cefotaxime and 100 mg/L kanamycin. Tomato shootswhich rooted in kanamycin after about 2-3 weeks were transferred to soilin 8" pots and covered with plastic bags. The plants were grown undermixed fluorescent and incandescent lights for a 12 hr, 24° C. day; 12hr, 20° C. night cycle, with approximately 80% relative humidity, forone week before removing the plastic bags. The plants were grown foranother 2-4 weeks before performing ALS assays. An increase ofuninhibited ALS activity in the presence of the sulfonylurea Classic® inleaf extracts from transformed plants was demonstrated in theseexperiments (Table 11).

                  TABLE 11                                                        ______________________________________                                        ALS Activity of Wild-Type and Transformed Tomato                                       Percent Uninhibited ALS Activity.sup.1                                        0 ppb  10 ppb   100 ppb   1000 ppb                                   ______________________________________                                        Wild-type  100      15        5       4                                       Transformant #3                                                                          100      42       25      12                                       Transformant #4a                                                                         100      60       42      26                                       Transformant #4b                                                                         100      29       15       5                                       Transformant #4c                                                                         100      58       43      25                                       Transformant #4d                                                                         100      29       15      10                                       ______________________________________                                         .sup.1 The ALS activities in each line are relative to the activity in th     absence of herbicide which is taken as 100 percent. The sulfonylurea          compound used was DPXF6025. the active ingredient in Classic ®            herbicide.                                                               

The assay for ALS activity in the absence or presence of herbicide fromtransformed or untransformed plants was conducted as follows:

1. Grind 2.5 grams of relatively young leaf tissue (4-6 inches inlength) in a tissue homogenizer containing 10 ml extraction buffer (100mM KHP0₄ pH 7.5, 0.5 mM MgCl2, 10% glycerol, 1 mM pyruvate, 0.5 mM TPP,10 nM FAD) and 200 mg Polyclar AT (BDH Biochemicals). Keep on ice.

2. Homogenize extract for approximately 10 seconds in a polytron(Brinkman Instruments) on setting #7.

3. Centrifuge extract in a Sorvall BS-34 rotor, 20 min. 16K rpm, 4° C.

4. Equilibrate PD-10 (Pharmacia) columns by washing with column buffer(100 mM KHP04 pH 7.5, 0.5 mM MgCl₂, 10% glycerol, 1 mM pyruvate) 5times.

5. To plant extract supernatant, add cold saturated (NH4)₂ SO₄ toachieve a 50% cut. Incubate on ice for 30 minutes.

6. Centrifuge extract in SS-34 rotor, 20 minutes, 16K rpm, 4° C. Decantsupernatant.

7. Resuspend pellet in 1 ml cold column buffer.

8. Load extract onto column and chase with a volume of column buffer toachieve total volume loaded equal to 2.5 ml. Let this run throughcolumn.

9. Elute proteins With 2× volume of extract loaded. Recover in 15 mlFalcon tube placed beneath column.

10. Set up reaction for each extract in microfuge tubes as follows: 350μl reaction mix (200 mM pyruvate, 5 mM TPP, 0.9 mM FAD, 5 mM KHPO₄ pH7.0), 50 μl of either 5 mM KHPO₄ or desired sulfonylurea concentration,and 100 μl plant extract.

11. Incubate reaction for 1 hour, 30° C.

12. To stop reaction, add 50 μl M 6M H₂ SO₄ and incubate at 60° C. for10 minutes. Spin in microfuge 5 minutes.

13. Set up color development tubes as follows: 500 μl 0.5% creatin,reaction tube supernatant, 0.5 ml α-napthol solution (1.5 g α-napthol in30 ml 2.5 N NaOH). Mix and heat at 60° C. for 20 minutes.

14. Vortex each tube and load 100 μl of each sample into wells ofmicrotiter plate. Read at OD 530.

    ______________________________________                                        Callus Induction Medium                                                       1 package of MS salts (Gibco                                                                           per liter                                            Murashige Organics Medium with                                                                         pH 5.8                                               3% sucrose                                                                    1 ml of 1 mg/ml NAA                                                           0.2 ml of 1 mg/ml BAP                                                         0.8% agar                                                                     CN                                                                            Shoot Induction Medium                                                        1 package of MS salts with 3% sucrose                                                                  per liter                                            1 ml of 1 mg/ml NAA      pH 5.8                                               1 ml of 1 mg/ml BAP                                                           0.8% agar                                                                     A                                                                             Root Induction Medium                                                         1 package of MS salts (without sucrose)                                                                per liter                                            10 grams sucrose         pH 5.8                                               0.8% agar                                                                     Agrobacterium R-Medium                                                        Add 7.5 g agar to 440 ml H.sub.2 O, autoclave, and keep at                    55° C. Add sterile stocks of:                                          0.5      Ml          1 M MgSO.sub.4                                           0.5      ml          1 M CaCl.sub.2                                           10.0     ml          20% sucrose                                              5.0      ml          100 mg/ml kanamycin                                      50.0     ml          10x salts (Na.sub.2 HPO.sub.4.7H.sub.2 O                                      60 g/l;                                                                       KH.sub.2 PO.sub.4, 30 g/l; NaCl,                                              5 g/l,                                                                        NH.sub.4 Cl, 10 g/l)                                     CTM Medium                                                                    1 pkg MS salts                                                                1 ml B5 vitamins                                                                           (per 100 ml; Nicotinic Acid 100 mg,                                           thiamine, hydrochloride 1000 mg, pyri-                                        doxine hydrochloride 100 mg,                                                  M-inositol 10,000 mg)                                            3 mM MES                                                                      3% glucose                                                                    0.7% agar                                                                     pH 5.7                                                                        Autoclave and add 1 ml 1 mg/ml zeatin stock                                   OMS Medium                                                                    1 pkg MS salts                                                                1 ml B5 vitamins (see above)                                                  3 mM MES                                                                      3% sucrose                                                                    0.8% agar                                                                     pH 5.7                                                                        Min A + Tetracycline (1 ug/ml) Medium                                         1.     Add 7.5 g agar to 400 ml H.sub.2 O                                     2.     Make stock:                                                                   K.sub.2 HPO.sub.4        5.25   g                                             KH.sub.2 PO.sub.4        2.25   g                                             (NH.sub.4).sub.2 SO.sub.4                                                                              0.5    g                                             Sodium Citrate 2H.sub.2 O                                                                              0.25   g                                                                      100    ml                                     3.     Make MgSO.sub.4 . 7H.sub.2 O stock = 20 g/100 ml,                             autoclaved                                                             4.     Make glucose stock = 20% solution, autoclaved                          5.     Make tetracycline stock = 1.0 mg/ml in                                        ethanol/H.sub.2 O, 50% v/v filter sterilized                           To make Min A medium + 1 μg/ml tetracyline:                                       Mix (1) and (2)                                                               Add 0.5 ml of (3), 5 ml of (4). and 0.5 ml of (5)                      YEB Medium                                                                                              per liter                                                  Bacto Beef Extract       5.0    g                                             Bacto Yeast Extract      1.0    g                                             Peptone                  5.0    g                                             Sucrose                  5.0    g                                             MgSO.sub.4 .7H.sub.2 O   0.5    g                                             Agar (optional)          15.0   g                                      Herbicide solutions:                                                                         A 1 ppm stock solution of sulfonyl-                                           urea herbicide can be made by                                                 dissolving 1 mg of herbicide in 100                                           ml of 0.01N NH.sub.4 OH, and then                                             diluting 1:10 with 5 mM KHPO.sub.4 pH                                         7.0. This stock will suffice to                                               assay herbicide concentrations of                                             100 ppb or lower. If higher                                                   concentrations are desired,                                                   dissolve 1 mg in 10 ml of 0.01N                                               NH.sub.4 OH. etc.                                              Herbicide dilutions:                                                                         In the standard assay, 50 μl of                                            herbicide is added to 450 μl                                               assay mix and extract, for a 1:10                                             dilution of herbicide. So, for                                                each concentration to be tested, a                                            10× solution in 5 mM KHPO.sub.4 pH 7.0                                  should be diluted from the stock                                              solution.                                                      ______________________________________                                    

EXAMPLE VII

The tobacco SURB-Hra gene encoding herbicide resistant ALS was used totransform Beta vulgaris (sugarbeet) to herbicide resistance by thefollowing Agrobacterium tumefaciens mediated transformation procedure.

In order to surface sterilize seeds, 50-100 seeds were placed in a25×100mm sterile petri dish in a laminar flow hood and 25-30 ml of 70%ethanol was added. Seeds were agitated by hand 1-2 min., the ethanol wasdecanted, and 25-30 ml 20% Clorox (20 ml commercial bleach/80 ml sterileH20/1 drop Tween 80) was added. The seeds were agitated on a gyrotaryshaker at 40 rpm for 20 mins., and the bleach was decanted. The bleachsterilization was repeated 2 times for a total of 60 min., and thesterilized seeds were rinsed 3 times for 5 min. each with 25-30 mlsterile H₂ O.

To germinate the seeds, they were plated on 1/2PG_(O) agar solidifiedmedium, 12 seeds/50 ml media/15 mm×150 mm petri dish, and cultured at24° C. in darkness.

NOTE: 10-20% contamination of seed may be anticipated for a good, cleanseed lot. For this reason seed is plated far apart on large plates andthe germinations are monitored continuously and non-contaminated seedare transferred to fresh plates. If contamination is fungal, thentransfers are conducted in a laminar flow cabinet with the fan off.

60-80% germination is expected for a good seed lot. Germination is notsynchronous. An approximately 14 day period is required to obtain (a)all germinations and (b) sufficient elongation to provide manyappropriate explants. Agrobacterium overnight (O/N) suspension cultureswere prepared as described in EXAMPLE I. Freshly plated Agrobacteriumhave shorter lag times than cultures stored for long periods at 5° C. Itis important that log phase bacteria be used as inocula. Therefore, atrial should be conducted to assure an overnight culture which reacheslog phase (OD 550mm 0.4-0.8) before hypocotyl inoculation.

To prepare plant explants, hypocotyl were cut into approximately 0.5cmsections with a sharp surgical blade, and plated immediately onto agarsolidified PG_(O) ⁰.1 /0.1. Do not allow dessication, nor damage thewound by the use of a dull blade or by compressing with forceps.

To inoculate explants, they were dipped individually into a log phasesuspension of the appropriate Agrobacterium strain. They were immersedbriefly (1-3 sec.) and arranged in a grid pattern on a fresh plate: 25explants/100 mm plate of agar solidified PG_(O) ⁰.1 /0.1.

The explants were dried by leaving plates open in a laminar flow hood10-30 min. This concentrates Agrobacteria onto the wound. It also maylimit possible damage by water logging. It is important, though, not todessicate tissue. Close observation of this step is required. The plateswere then sealed with parafilm and cultured at 24° C. for 3 days.

The explants were collected into liquid PG ⁰.1 /0.1 containingcefotaxime 500 μg/ml in a 25×100 mm petri dish, and shaken gently on agyrotary shaker at 40 rpm for 1 hr. The media was decanted, replacedwith fresh counterselective media, and shaken gently for an additional 2hrs. The explants were plated in grids, 25/100 mm plate agar solidifiedPG ⁰.1 /0.1 containing cefotaxime 500 μg/mL and cultured at 24° C. for 3days.

Selection for transformed plant cells was applied as follows. Explantswere transferred to fresh plates of agar solidified PG ⁰.1 /0.1containing vancomycin 100μg/ml or chlorsulfuron, 2 ng/ml as selectiveagents. The number of resistant colonies was counted after 20-30 days.More than one may be observed at each wound. Transformants were excisedas follows. Callus was removed from the wound/explant with a surgicalblade and cultured independently on fresh selective media. SomeAgrobacterium can escape from counter-selection. Additional washes incefotaxime containing liquid media are possible as is repeated transferto cefotaxime containing agar solidified plates. Under the suggestedprotocol we observed approximately 15% explant contamination, which wasan acceptable loss. The results of a transformation experiment using thesugarbeet line Beta vulgaris 87193 are shown in Table 12. The level ofchlorsulfuron resistance in calli of B. vulgaris transformed with theSURB-Hra mutant ALS gene of tobacco, is compared to that ofuntransformed calli. These results demonstrate that the tobacco SURB-Hragene, encoding herbicide-resistant ALS, can be expressed efficiently insugarbeet.

In another experiment, sugarbeet cells were transformed to herbicideresistance with the tobacco SURB-Hra gene and the in vitro constructedsite-specific mutations made in the sugarbeet ALS gene (see Example IVfor construction of sugarbeet ALS gene mutations). The results shown inTable 13 indicate that in vitro constructed mutant sugarbeet ALS genescan be used to transform efficiently sugarbeet cells to herbicideresistance. The selective resistance mutation, Ala (122)-Pro, did notyield chlorsulfuron resistant transformants, as expected.

                                      TABLE 12                                    __________________________________________________________________________    Growth of Sugarbeet Calli Tansformed With                                     a Tobacco SURB-Hra ALS Gene in the                                            Presence of Increasing Concentrations of Chlorsulfuron                        Chlorsulfuron (ppb)                                                                     0   10  30  100 300 1000                                                                              3000                                                                             10000                                    __________________________________________________________________________    87193/152.sup.1                                                                         15/15                                                                             15/15                                                                             15/15                                                                             15/15                                                                             15/15                                                                             15/15                                                                             3/15                                                                             0/15                                     87193/0.sup.2                                                                           15/15                                                                              0/15                                                                              0/15                                                                             nd  nd  nd  nd nd                                       __________________________________________________________________________     .sup.1 Beta vulgaris line 87193 transformed with Agrobacterium tumefacien     LBA4404 carrying plasmid pAGS152.                                             Untransformed Beta vultaris line 87193.                                  

                                      TABLE 13                                    __________________________________________________________________________    Herbicide Resistance in Sugarbeet Cells                                       Transformed with Site Specific Mutant                                         Sugarbeet ALS Genes                                                           Gene Origin                                                                            Tobacco                                                                             Sugarbeet                                                                           Sugarbeet                                                                           Sugarbeet                                                                           Sugarbeet                                    __________________________________________________________________________    Mutation pro(197)-                                                                           pro(197)-                                                                           trp(591)-                                                                           pro(197)-                                                                           ala(122)-                                             ala/trp                                                                             ala/trp                                                                             leu   ala   pro                                                   (591)-leu                                                                           (591)-leu                                                      Chlorsulfuron.sup.R                                                                    47    36    20    24     0                                           Hygromycin.sup.R                                                                       93    97    95    98    98                                           Chlorsulfuron.sup.R /                                                                  .51   .37   .21   .24   --                                           Hygromycin.sup.R                                                              __________________________________________________________________________     Gene introductions were done by standard hypocotyl inocculation protocol.     For each construction 100 hypocotyl wounds were cocultured and selected o     chlorsulfron at 10 μg/ml and 100 wounds were cocultured and selected o     hygromycin at 10 ng/ml. The number of wound sites which developed             resistant callus was counted 60 days after innoculation.                 

    __________________________________________________________________________    Media and Amendments                                                                       Ingredient  Stock                                                                              (Final)                                                                             Amt./Liter                                __________________________________________________________________________    1/2 PG.sub.O PG.sub.O Majors A                                                                         10X        50 ml                                                  PG.sub.O Majors B                                                                         100X       5                                                      FeEDTA      100X       5                                                      B5 vitamins 100X       5                                                      MS micronutrients                                                                         1000X      1                                                      Sucrose          1.5% w/v                                                                            15 gm                                                  Mes buffer       3 mM  590 mg                                                 T.C. agar        0.8% w/v                                                                            8 gm                                                   pH 5.7, autoclave sterile, dispense                                           aspectically into 15 × 150 mm petri plates.                PG.sub.O .sup.0.1 /.sub.0.1                                                                PG.sub.O Majors A                                                                         10X        100 ml                                                 PG.sub.O Majors B                                                                         100X       10                                                     FeEDTA      100X       10                                                     B5 vitamins 100X       10                                                     MS micronutrients                                                                         1000X      1                                                      Sucrose          3.0% w/v                                                                            30 gm                                                  Mes buffer       3 mM  590 mg                                                 24-D        1 mg/ml    100 μl                                              B1AP        1 mg/ml    100 μl                                              pH 5.7, autoclave sterile.                                       PG.sub.O .sup.0.1 /.sub.0.1                                                                agar solidified, as above except with T.C.                                    agar 0.8% w/v. Dispense 25 ml/25 × 100                                  petri dish.                                                      __________________________________________________________________________    Stock Solutions                                                                                                   Amount/                                   Stock        Ingredient  MW   (Final)                                                                             Liter                                     __________________________________________________________________________    PG.sub.O Majors A (10X)                                                                    KNO.sub.3   101.1                                                                              19.8 mM                                                                             20                                                     (NH.sub.4).sub.2 SO.sub.4                                                                 132.14                                                                             3     4                                                      KCl         74.55                                                                              8     6                                                      MgSO.sub.4.7H.sub.2 O                                                                     146.5                                                                              2     5                                                      CaCl.sub.2.2H.sub.2 O                                                                     147.0                                                                              2     3                                         PG.sub.O Majors B (100X)                                                                   NaH.sub.2 PO.sub.4                                                                        120.0                                                                              2.1 mM                                                                              25                                        Fe EDTA (100X)M                                                                            FeSO.sub.4 7H.sub.2 O                                                                          100 μm                                                                           2.78 gm                                                Na.sub.2 EDTA    100 μm                                                                           3.72                                                   Dissolve EDTA first. pH 3.0. Store @ 4° C.,                            dark                                                             B5 vitamins (100X)                                                                         Nicotinic acid   1 mg/lit                                                                            0.1 gm                                                 Thiamine HCl     10    1.0                                                    Pyridoxine HCl   1     0.1                                                    Myo-inositol     100   10                                        MS micronutrients (1000X)                                                                  MnCl.sub.2 4H.sub.2 O                                                                     197.9                                                                              100 μm                                                                           19800 mg                                               H.sub.3 BO.sub.3                                                                          01.8 100   6200                                                   ZnSO.sub.4 7H.sub.2 O                                                                     287.5                                                                              30    8625                                                   KI          166  5     830                                                    NaMoO.sub.4 2H.sub.2 O                                                                    206  1.2   250                                                    CuSO.sub.4 5H.sub.2 O                                                                     249.7                                                                              0.1   25                                                     CoCl.sub.2 6H.sub.2 O                                                                     237.9                                                                              0.1   25                                                     Dissolve MnCl.sub.2 4H.sub.2 O in dil HCl                                     Dissolve one at a time and completely                                         before adding next.                                                           Boil, cool, pH 4.0, store dark at 4° C.                   __________________________________________________________________________

EXAMPLE VIII

The tobacco SURB-Hra gene encoding herbicide resistant ALS was used totransform Brassica napus cv. Olga by the following Agrobacteriumtumefaciens mediated transformation procedure.

To surface sterilize seeds, they were immersed for 1 min. in 70%ethanol, then 30-60 min. in Clorox Tween (see EXAMPLE VII). The surfacesterilized seeds were germinated on 1/2 MS, 1/2 PGO (SEE EXAMPLE VII),at 24° C. in the dark. At 5-10 days post germination, hypocotyls weredivided into 0.5 cm sections and placed on solid I medium containingacetosyringone 100 μm (Aldrich Chemical)(IAS100).

Immediately the explants were dipped individually into a log phasesuspension of LBA 4404 containing binary plasmid pAGS15.

The explants were plated onto IAS100. The Agrobacterium droplet wascarefully dried down onto the tissue by leaving the plate open in alaminar flow hood. Co-cultivation was conducted at 24° C. in low lightor darkness for 3 days.

After 3 days the explants were collected into liquid I medium containingcefotaxime 500 mg/L in 100×25 mm petri dishes, and shaken on a gyrotoryshaker at 40 rpm for 3 hrs.

The explants were plated on solid I medium containing cefotaxime 500mg/mL, and cultured for 3 days at 24° C. in low light.

The explants were plated on solid I medium containing vancomycin 100mg/L and kanamycin 100 mg/L.

After about 1 month transformed callus appeared as discreet nodules atthe ends of explants.

As nodules appeared, they were excised with a sharp scalpel and placedon solid I medium containing kanamycin 100 mg/L.

When transformed callus reached a sufficient size (0.5 cm diameter) itwas transferred to KR medium containing kanamycin 100 mg/L. Thismaterial regenerates fastest if it is plated on fresh media every twoweeks. Roots were regenerated on 1/2 MS containing IBA 2 μm.

In one experiment, of 100 wound sites (either end of 0.5 cm hypocotylsector) 20 developed callus tissue which was resistant to kanamycin (100mg/L). Five of the 20 transformed cell lines were subsequently inducedto regenerate on kanamycin and somatic siblings for each regenerantgenotype were produced by nodal multiplication. These plants weresprayed with various chlorsulfuron concentrations and the results aresummarized in Table 14. Two of the five transformants are resistant tochlorsulfuron at levels which are about 10 times greater than that whichis lethal to control (untransformed) plants.

                  TABLE 14                                                        ______________________________________                                                  0.3 ppm                                                                              1 ppm     3 ppm   10 ppm                                     ______________________________________                                        untransformed                                                                             ++       +         -     -                                        R.sub.o #1  ++       ++        ++    +                                        R.sub.o #2  ++       +         -     -                                        R.sub.o #3  ++       +         +     -                                        R.sub.o #4  ++       +         -     -                                        R.sub.o #5  +++      ++        ++    +                                        ______________________________________                                         +++ normal growth, no axial induction                                         ++ reduced growth, sublethal at apis, axial induction                         + reduced growth, lethal at apis, axial induction                             - lethal                                                                 

To further demonstrate the expression of the SURB-Hra gene intransformed Brassica napus, an ALS assay in the presence of theherbicide chlorsulfuron was performed as described in EXAMPLE VI. TheALS activities of the untransformed parent and transformant R_(o) #5(Table 14) were compared (Table 15). A consistent increase in thepercent uninhibited ALS activity was observed in the transformant. Thus,the tobacco SURB-Hra gene, encoding herbicide-resistant ALS, can beexpressed in Brassica napus, but that expression is not efficient.Addition of nucleotide regulatory sequences that provide higher levelexpression in Brassica napus would be expected to increase the level ofherbicide resistant ALS and the level of tolerance to foliarapplications of the herbicide.

                  TABLE 15                                                        ______________________________________                                        ALS Activity of Wild Type and                                                 Transformed Brassica napus                                                    ______________________________________                                                Percent Uninhibited ALS Activity.sup.1                                          0 ppb   1 ppb    10 ppb                                                                              100 ppb                                                                              1000 ppb                              ______________________________________                                        Wild Type 100.0   86.6     28.2  10.1    7.6                                  Transformant                                                                            100.0   88.1     36.6  20.1   14.5                                  R.sub.o #5                                                                    ______________________________________                                        Brassica napus       Culture Media                                                                               Amount/                                    Ingredient      Stock    (Final)   Liter                                      ______________________________________                                        I Media                                                                       MS Major Salts  10X                100  ml                                    MS Micronutrients                                                                             1000X              1    ml                                    Fe EDTA         100X               10   ml                                    I Vitamins      100X               10   ml                                    2,4-D           1 mg/ml            0.2  ml                                    Kinetin         1 mg/ml            3    ml                                    Sucrose                  3% w/v    30   gm                                    Mannitol                 1.8% w/v  18.2 gm                                    T.C. agar                0.8% w/v  8    gm                                    Mes Buffer               3 mM      0.59 gm                                                    pH 5.7, autoclave sterile                                     KR Media                                                                      K3 Major Salts  10X                100  ml                                    CaCl.sub.2 2H.sub.2 O                                                                         100X               10   ml                                    MS Micronutrients                                                                             1000x              1    ml                                    Fe EDTA         100X               10   ml                                    B5 Vitamins     100X               10   ml                                    Zeatin*         1 mg/ml            2    ml                                    IAA*            1 mg/ml            0.1  ml                                    Sucrose                  1% w/v    10   gm                                    Xylose                   0.025%    0.25 gm                                                             w/v                                                  Agarose (Type 1, low E/Eo)                                                                             0.25% w/v 2.5  gm                                    Mes Buffer               3 mM      0.59 gm                                                    pH 5.7, autoclave sterile                                     ______________________________________                                        Brassica napus  Stock Solutions                                               Stock   Ingredient  (Stock) (Final)                                                                              Amount/Liter                               ______________________________________                                        MS Major                                                                              NH.sub.4 NO.sub.3                                                                         10X     20.5 mM                                                                              16.5 gm                                    Salts   KNO.sub.3           18.8   19.0                                               MgSO.sub.4 7H.sub.2 O                                                                             1.5    3.7                                                KH.sub.2 PO.sub.4   1.25   1.7                                                CaCl.sub.2 2H.sub.2 O                                                                             3.0    4.4                                        K3 Major                                                                              KNO.sub.3   10X     25.0 mM                                                                              25.0 gm                                    Salts   (NH.sub.4).sub.2 SO.sub.4                                                                         1.0    1.34                                               MgSO.sub.4 7H.sub.2 O                                                                             1.0    2.5                                                KH.sub.2 PO.sub.4   1.5    2.01                                               NH.sub.4 NO.sub.3   3.1    2.5                                        CaCl.sub.2                                                                            CaCl.sub.2 2H.sub.2 O                                                                     100X    6.3 mM 92.3 gm                                    2H.sub.2 O                                                                    MS      MNCl.sub.2 4H.sub.2 O                                                                     1000X   100 μm                                                                            19800 mg                                   Micro-  H.sub.3 BO.sub.3    100    6200                                       nutrients                                                                             ZnSO.sub.4 7H.sub.2 O                                                                             30     8625                                               KI                  5      830                                                NaMoO.sub.4 2H.sub.2 O                                                                            1.2    250                                                CuSO.sub.4 5H.sub.2 O                                                                             0.1    25                                                 CoCl.sub.2 6 H.sub.2 O                                                                            0.1    25                                         Fe EDTA Na.sub.2 EDTA                                                                             100X    100 μm                                                                            3.73 gm                                            FeSO.sub.4 7H.sub.2 O                                                                             100    2.78                                       I Vitamins                                                                            Myo-Inositol                                                                              100X    100 mg/l                                                                             10000 mg                                           Thiamine            0.5    50                                                 Glycine             2.0    200                                                Nicotinic acid      5.0    500                                                Pyrodoxine          0.5    50                                                 Folic acid          0.5    50                                                 Biotin              0.05   5                                          ______________________________________                                         .sup.1 The ALS activities are relative to that in the absence of herbicid     which is taken as 100 percent. The sulfonylurea compound used was             chlorsulfuron (DPXW4189), the active ingredient in Glean ® herbicide.     *add these filter sterilized components aseptically                      

EXAMPLE IX

The tobacco SURB-Hra gene encoding herbicide resistant ALS was used totransform Cucumis melo cv. Amarillo Oro (melon) to herbicide resistanceby the following Agrobacterium tumefaciens mediated transformationprocedure. A reference to this procedure is Moreno, V., et al. Plantregeneration from calli of melon. Plant Cell Tissue Organ Culture, 5(1985) 139-146.

Surface sterilization of seeds was greatly facilitated by first removingthe seed coat. The seeds were then sterilized by rinsing in 70% ethanolfor 2 min., then washing in Clorox/Tween (see EXAMPLE VII) for 20 mins.The sterile seeds were washed 3 times in sterile distilled H₂ O andgerminated on OMS at 24° C. with a 16 hr. day length.

Cotyledons of 7-14 day old melon seedlings were cut into 5 mm sliceswith a fresh, sharp scalpel. These explants were dipped into a log phaseAgrobacterium culture prepared as described in EXAMPLE I, transferred tofresh co-cultivation plates and cultured at 24° C. with 16 hr. days for3 days.

The bacteria were killed by washing the explants for 3 hrs. with gentleagitation in washing media and cultured on fresh selection plates.

The explants were subcultured every 3-4 weeks, dissecting the morecompact, darker green sectors away from the white fluffier callus.

When "morphogenic" callus (very dark green, compact, perhaps somerecognizable leaves) was seen, it was transferred to regeneration media.The tissue can go directly to shoots instead of going through themorphogenic stage. Shoots were rooted in rooting media. Approximately70% of the explants developed callus resistant to kanamycin at 100 μm/1.Transformed callus was put on media containing increasing concentrationsof chlorsulfuron and growth in the presence of herbicide was determinedby weighing the callus after 30 days (Table 16). Some transformants grewas well at high concentrations of chlorsulfuron (1000 ppb) as in itsabsence, e.g. Trans 1, Trans 2 and Trans 7. Thus the tobacco SURB-Hragene can function to transform melon to high level herbicide resistance.

                  TABLE 16                                                        ______________________________________                                                Non-                                                                  Chlorsul-                                                                             trans-                                                                furon ppb                                                                             formed  Trans 1 Trans 2                                                                             Trans 3                                                                             Trans 4                                                                             Trans 5                             ______________________________________                                          0     5.7     30.4    30.4  30.4  30.4  30.4                                 50     0       44.5    19.5  10    14    175                                  100    0       25.9    0     7.5   16.6  70                                   500    0       51.9    14.8  0     0     3.7                                 1000    0       46      26    11.7  10    5.7                                 2000    0       19.1    8     28.7  5     0                                   3000    0       15.2    18    0     0     0                                   4000    0       41.9    3.3   0     0     0                                   5000    0       14.4    0     3.6   0     0                                   ______________________________________                                                        Trans 6 Trans 7                                                                             Trans 8                                                                             Trans 9                                                                             Trans 10                            ______________________________________                                                        30.4    26.4  28.3  27.2  40                                                  28.8    46.5  18.1  27.3  40                                                  18.3    25.5  14.9  11.1  39                                                  2       16.1  2     10.4  18.6                                                4.7     19.3  2.6   9     27.6                                                0       19.3  0     8.8   23.5                                                0       17.1  10    13.7  17.6                                                0       20.7  3.4   3     7.6                                                 0       26.5  7.4   2.6   8.9                                 ______________________________________                                        Measurements indicate fold increase in weight of callus.                      ______________________________________                                        Media                                                                         OMS                                                                           MS Salts and Fe EDTA   1X                                                     B5 Vitamins            1X                                                     Sucrose                3%                                                     MES                    3 mM                                                   pH 5.7                                                                        T.C. agar              0.8%                                                   Autoclave 20 min.                                                             Basic medium                                                                  MS Salts and Fe EDTA   1X                                                     Myo inositol           100 mg/l                                               Thiamine               1 mg/l                                                 Sucrose                3%                                                     MES                    3 mM                                                   pH 5.7                                                                        T.C. agar              0.8%                                                   Autoclave 20 min.                                                             Co-cultivation Medium is Basic medium plus:                                   Acetosyringone 100 μm (acetosyringone                                      is kept as a 100 mm stock in DMSO)                                            Kinetin               6      mg/l                                             IAA                   1.5 mg/l                                                Washing Medium is Basic Medium without agar plus:                             Cefotaxime            500    mg/l                                             Kinetin               6 mg/l                                                  IAA                   1.5 mg/l                                                Selection Medium is Basic Medium plus:                                        Kinetin               6      mg/l                                             IAA                   1.5 mg/l                                                Vancomycin            100 mg/l                                                One of the following selective drugs                                          depending upon Agrobacterium                                                  construction:                                                                 Kanamycin             100    mg/l                                             Hygromycin            50 mg/l                                                 Chlorsulfuron         100 mg/l                                                Regeneration Medium is Basic Medium plus:                                     BAP                   0.1    mg/l                                             Vancomycin            100 mg/l                                                Selective drugs as above                                                      Rooting Medium is OMS plus:                                                   IBA                   2      μm                                            Vancomycin            100    mg/l                                             Selective drugs as above                                                      ______________________________________                                    

EXAMPLE X

The tobacco SURB-Hra gene encoding herbicide resistant ALS was used totransform Medicago sativa cv. Rangelander (alfalfa) to herbicideresistance by the following Agrobacterium tumefaciens mediatedtransformation procedure. A reference to this procedure is Deak, M.,Kiss, G., Koncz, C., and Dudits, D. Transformation of Medicago byAgrobacterium mediated gene transfer (preprint).

Plants were grown and subcultured in OMS. The materials used werepetioles and stem segments (5 mm in length) from plants about 2 monthsfrom the last subculture.

The petioles and stems of sterile plants were cut into 5 mm lengths witha fresh, sharp scalpel. These explants were dipped into a log phaseAgrobacterium culture prepared as described in EXAMPLE I, transferred tofresh co-cultivation plates, and cultured at 24° C. with 16 hr. days for3 days.

The bacteria were killed by washing the explants for 3 hrs. with gentleagitation in washing media, and cultured on fresh selection plates.

The explants were subcultured every 3-4 weeks. In about 1 monthtransformed callus growing out of the wounded ends of the explants wasseen, dissected away from the explant and plated on fresh selectionmedia. When callus became more organized, (areas are darker green andmore compact) it was transferred to fresh maturation media.

When developed embryos appeared, they were transferred to germinationmedia. After germination, the small plants were grown on the samemedium.

Less than 1% of explants developed callus resistant to kanamycin at 100mg/L. Kanamycin resistant sectors were found to be resistant to theherbicide chlorsulfuron at 50 ppb. Three shoots were produced fromkanamycin resistant callus. Tissue from these transformants was assessedfor herbicide resistant growth over a range of chlorsulfuronconcentrations. One transformant was able to grow at 1000 ppbchlorsulfuron; the other two were able to grow at 5000 ppb. Thus, thetobacco SURB-Hra gene can function to transform alfalfa to high levelherbicide resistance.

    ______________________________________                                        Media                                                                         OMS                                                                           MS Salts and Fe EDTA      1X                                                  B5 Vitamins               1X                                                  Sucrose                   3%                                                  MES                       3 mM                                                pH 5.7                                                                        T.C. agar                 0.8%                                                Autoclave 20 min                                                              Basic Medium                                                                  MS Salts and Fe EDTA      1X                                                  UM Vitamins               1X*                                                 Sucrose                   3%                                                  pH 5.7                                                                        T.C. agar                 0.8%                                                Autoclave 20 min.                                                             *100X UM vitamins                                                             (amounts given for 100 ml of 100x stock)                                      Thiamine HCl              1 g                                                 Nicotinic acid            0.5 g                                               Pyridoxine HCl            1 g                                                 Myo inositol              10 g                                                Glycine                   0.2 g                                               Co-Cultivation Medium                                                         Basic Medium plus                                                             Acetosyringone 100 μm (Acetosyringone                                      is kept as a stock of 100 mM in DMSO)                                         2.4-D                     0.5 mg/l                                            BAP                       0.2 mg/l                                            Washing Medium                                                                Basic Medium without agar plus                                                Cefotaxime                500 mg/l                                            2,4-D                     0.5 mg/l                                            BAP                       0.2 mg/l                                            Selection Medium                                                              Basic Medium plus                                                             2,4-D                     0.5 mg/l                                            BAP                       0.2 mg/l                                            Vancomycin                100 mg/l                                            One of the following selective drugs,                                         depending upon Agrobacterium                                                  construction:                                                                 Hygromycin                50 mg/l                                             Chlorsulfuron             100 mg/l                                            Maturation Medium                                                             Same as selection medium without 2,4-D                                        Germination Medium                                                            Basic Medium plus                                                             Vancomycin                100 mg/l                                            Selective drugs as above                                                      ______________________________________                                    

EXAMPLE XI

Sulfonylurea herbicide-resistant mutants of Arabidopsis thaliana (L.)Heynh were isolated as follows: M1 plants derived from approximately100,000 ethyl methane sulfonate-mutagenized wild type seeds wereself-fertilized. Approximately 30,000 of the resultant M2 seeds wereplaced on a medium containing 200 nM (75 ppb) chlorsulfuron, aconcentration that completely inhibits germination of the wild typeseeds. Four seeds germinated in the presence of chlorsulfuron. Theherbicide resistance trait in one mutant, designated GH50, was shown tobe stably inherited and to be due to a single, dominant nuclearmutation. Plants carrying the mutation were resistant to concentrationsof chlorsulfuron and sulfometuron methyl that were at least 100-fold and10-fold, respectively, higher than that required to inhibit wild typeplants. The concentrations of chlorsulfuron and sulfometuron methylrequired to inhibit 50% of the in vitro ALS activity in leaf extracts ofthe mutant were 1000-fold and 100-fold, respectively, greater than thatrequired for the wild type.

A genomic library of DNA from Arabidopsis which was homozygous for theherbicide resistance mutation of GH50 was made in bacteriophage lambda,and was screened for recombinant clones which hybridized to thepreviously isolated gene encoding a herbicide-sensitive ALS from wildtype Arabidopsis. A phage clone was identified which contained a 6.1kilobase Xba I DNA fragment that hybridized to the wild type ALS gene.This Xba I fragment was isolated and inserted into the Xba I site ofplasmid pKNKX. Plasmid pKNKX contains a bacterial gene NPT II, thatencodes neomycin phosphotransferase, fused to regulatory signals, thenopaline synthase promoter (NOSP) and transcription terminator, allowingexpression in plants and resulting in kanamycin resistance. PlasmidpKNKX was constructed as follows:

The precursor vector pKNK was derived from the commonly used plasmidpBR322 by removing the Hind III and BamH I sites and inserting at theCla I site an approximately 2.3 kb Cla I fragment which incorporated (a)a 320 bp Cla I-Bgl II sequence containing the promoter region of theneomycin phosphotransferase (NPT II) gene of transposon Tn 5 derived bythe conversion of a Hind III site to the Cla I site [Beck, E., Ludwig,G., Auerswald, E. A., Reiss, B. & Schaller, H. (1982) Gene 19:327-336],(b) a 296 bp Sau 3A-Pst I sequence containing the nopaline synthasepromoter derived from the nopaline synthase gene (NOS) (nucleotides -263to +33, with respect to the transcription start site [Depicker, A.,Stachel, S., Dhaese, P., Zambryski, P & Goodman, H. J. (1982) J. Mol.Appl. Genet. 1:561-574] by the creation of a Pst I site at theinitiation codon, (c) the 998 bp Hind III- BamH I sequence containingthe coding sequence for the NPT II gene derived from Tn 5 by thecreation of Hind III and BamH I sites at nucleotides 1540 and 2518[Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B. & Schaller, H. (1982)Gene 19:327-336], respectively, and (d) the 702 bp BamH I-Cla I sequencecontaining the 3' region of the NOS gene (nucleotides 848 to 1550)[Depicker, A., Stachel, S., Dhaese, P., Zambryski, P. & Goodman, H. J.(1982) J. Mol. Appl. Genet. 1:561-574]. The nucleotide sequence at thefusion of the NOSP and the NPT II coding sequence is ##STR11##

Plasmid pKNK was, sequentially, linearized by digestion with restrictionenzyme Sal I, its ends made blunt by E. coli DNA polymerase I, Klenowfragment, and joined in the presence of T4 DNA ligase to phosphorylatedXba I linkers (5'-CTCTAGAG-3'). The excess linkers were removed by Xba Idigestion, followed by agarose gel electrophoresis. The linear plasmidDNA was isolated by electroelution, purified through a NACS column (BRL)and self-ligated in the presence of T4 DNA ligase. Ligated DNA was usedto transform competent E. coli HB101 cells. Ampicillin-resistant cellswere shown to contain plasmid pKNKX, which is identical to pKNK exceptfor the addition of an Xba I site next to the Sal I site.

Plasmid pKNKX was sequentially linearized with restriction enzyme Xba I,dephosphorylated with calf intestine phosphatase, phenol extracted, andjoined in the presence of T4 DNA ligase to an approximately 6.1 kb Xba Ifragment containing the mutant Arabidopsis ALS gene. The resultingplasmid, pKNKAR, has the open reading frames of the ALS gene and theNOS:NPT II gene in the vector in the same orientation. The Xba I insertis flanked by Sal I sites.

Plasmid pKNKAR was partially digested by restriction enzyme Sal I and,following phenol extraction, joined to a 1.25 kb Sal I fragmentcontaining the bacterial NPT I gene for bacterial kanamycin selectablemarker. The ligated molecules were used to transform competent E. coliHB101 cells and a kanamycin-resistant colony was shown to contain therecombinant plasmid pKAR (FIG. 9), in which the NPTI fragment wasinserted in the Sal1 site proximal to the ampicillin-resistance gene onthe vector.

Plasmid pKAR was conjugated into Agrobacterium tumefaciens bytriparental mating. Three ml overnight cultures of E. coli HB101 (pKAR)and E. coli HB101 (pRK2013) (ATCC number 37159) in LB liquid mediumcontaining 25 mg/L kanamycin were grown at 37° C., and of Agrobacteriumtumefaciens GV3850 in LB medium were grown at 28°-29° C. The cells wereharvested at room temperature in a clinical centrifuge, washed once inLB medium without drug, harvested, and resuspended in 3 ml of LB. 0.25ml aliquots of all three strains were mixed in a tube and the mixturewas transferred onto a Millipore filter (2.5 cm HAWP, 0.45 μm) placed ontop of three Whatman No. 1 filters in a petri dish. After all of theliquid medium was absorbed by the Whatman filter (about 30 min), theMillipore filter with bacteria on its top surface was laid (bacteriaside up) onto a LB plate without drug. After incubation overnight at28°-29° C., the Millipore filter was transferred to 5 ml of 10 mM MgSO₄and vortexed to resuspend the bacteria in the solution. 0.1 ml aliquotswere plated on selective plates [M9 minimal plates containing 20%sucrose, 1 mM MgSO₄, 1 mM CaCl₂, and 1 mg/ml kanamycin (Sigma)]. Severallarge colonies showed up after about four days of incubation at 28°-29°C. Several transconjugants were purified by three successivesingle-colony streakings on the same selective plates. Only Agrobacteriacontaining the plasmid pKAR recombined with the endogenous pGV3850plasmid through their common pBR322 sequences were expected to grow.This was confirmed by Southern analysis before using the engineeredAgrobacterium for plant transformations.

Another transconjugant GVKAS was made which was essentially identical toGVKAR, except that it had the 6.1 kb Xba I fragment derived from theherbicide-sensitive Arabidopsis thaliana.

For plant cell transformations, standard aseptic techniques for themanipulation of sterile media and axenic plant/bacterial cultures werefollowed, including the use of a laminar flow hood for all transfers.Recipes for media are given in Example VI. Potted tobacco plants forleaf disk infections were grown in a growth chamber maintained for a 12hr, 24° C. day, 12 hr, 20° C. night cycle, with approximately 80%relative humidity, under mixed cool white fluorescent and incandescentlights. Tobacco leaf disk infections were carried out essentially by themethod of Horsch et al. (1985) Science 227, 1229.

Young leaves, not fully expanded and approximately 4-6 inches in length,were harvested with a scalpel from approximately 4-6 week old tobaccoplants (Nicotiana tabacum var. Xanthi). The leaves were surfacesterilized for 30 minutes by submerging them in approximately 500 ml ofa 10% Chlorox, 0.1% SDS solution and then rinsed 3 times with steriledeionized water. Leaf disks, 6 mm in diameter, were prepared from wholeleaves using a sterile paper punch.

Leaf disks were inoculated by submerging them for several minutes in 20ml of a 1:10 dilution of an overnight Agrobacterium culture carrying thedesired plasmid. Agrobacterium cultures were started by inoculating 10ml of YEB broth with a single bacterial colony removed from an R-agarplate. The culture was grown for approximately 17-20 hours in 18 mmglass culture tubes in a New Brunswick platform shaker maintained at 28°C.

After inoculation, the leaf disks were placed in petri dishes containingCN agar medium. The dishes were sealed with parafilm and incubated undermixed fluorescent and "Gro and Sho" plant lights (General Electric) for2-3 days in a culture room maintained at approximately 25° C.

To rid the leaf risks of Agrobacterium and to select for the growth oftransformed tobacco cells, the leaf disks were transferred to fresh CNmedium containing 500 mg/L cefotaxime and 100 mg/L kanamycin. Cefotaximewas kept as a frozen 100 mg/ml stock solution and added aseptically(filter sterilized through a 0.45 μm filter) to the media afterautoclaving. A fresh kanamycin stock (50 mg/ml) was made for each useand was filter sterilized into the autoclaved media.

Leaf disks were incubated under the growth conditions described abovefor 3 weeks and then transferred to fresh media of the same composition.Shoots which developed on kanamycin-selected explants were excised andplaced in rooting medium A containing 100 mg/L kanamycin. After twoweeks, several small leaves were excised from each shoot, sliced into2-3 mm pieces, and placed on callus induction medium B containing either50 mg/L kanamycin, 10 ppb chlorsulfuron, or no selective agent. Callusformation was scored after three weeks of growth at approximately 25° C.on a 12-hour light/12-hour dark cycle. Seventeen of 19 transformantswhich received the sulfonylurea-resistant Arabidopsis ALS gene formedsecondary callus on 10 ppb chlorsulfuron: none of 22 transformants whichreceived the sulfonylurea-sensitive Arabidopsis ALS gene formed calluson the same medium.

Callus lines derived from transformed shoots were subcultured severaltimes to generate quantities of relatively uniform callus. Growthresponses to chlorsulfuron of these cell lines were tested by spreadingapproximately 50 mg of tissue on sterile double paper filter disks(Whatman #1) placed on the surface of callus medium B containing aseries of chlorsulfuron concentrations. Cultures were incubated for twoweeks under the conditions described above and then tissue was scrapedfrom each filter and weighed. Means and standard errors of means werecalculated from eight replicates for each cell line on each herbicideconcentration. Tobacco callus lines derived from transformants whichreceived the sulfonylurea-resistant Arabidopsis ALS gene were able togrow on chlorsulfuron concentrations 100 to 300 times higher than thosetolerated by lines derived from transformants which received the geneencoding sulfonylurea-sensitive ALS (Table 17).

Plants confirmed as transformants by rooting in 100 mg/L kanamycin andby secondary leaf callus formation on 50 mg/L kanamycin weretransplanted to soil. After 25-40 days of growth, extracts were preparedfrom two or three young, expanding leaves per plant and assayed for ALSactivity. For each extract, reactions containing either no herbicide or100 ppb chlorsulfuron were sampled at 10, 20, 30 and 40 minutes and thedata used to calculate rate of product formation. ALS extracted fromtransformants which received the sulfonylurea-resistant ALS gene wasinhibited 32-60% under these reaction conditions, while enzyme extractedfrom transformants which received the sulfonylurea-sensitive ALS genewas inhibited 94-97% (Table 18).

The plants assayed above were forced to self-pollinate by placing paperbags over immature flower heads. Seeds derived from theseself-pollinations were surface sterilized by stirring for 30 minutes in10% Chlorox, 0.1% SDS, rinsed three times in sterile deionized water,and plated on MMO medium containing a series of chlorsulfuronconcentrations. Progeny of transformants which received thesulfonylurea-resistant ALS gene showed simple Mendelian inheritance forthe ability to germinate and grow on chlorsulfuron concentrations ashigh as 3000 ppb, while progeny of transformants which received thesulfonylurea-sensitive ALS gene failed to survive on 30 ppbchlorsulfuron (Table 19).

                  TABLE 17                                                        ______________________________________                                        Growth Responses to Chlorsulfuron of Tobacco Callus Lines                     Derived From Transformants Which Received                                     Arabidopsis ALS Genes                                                                 Means and Standard Errors of Means                                            Calculated from Eight Replicates                                                Chlorsulfuron                                                       Callus    Concentration                                                                              Weight    % Uninhibited                                Line      (ppb)        (mg)      Growth                                       ______________________________________                                        GVKAS.sup.1 #4                                                                          0            2748 ± 202                                                                           100                                                    0.1          1576 ± 123                                                                           57.0                                                   0.3           452 ± 26                                                                            16.4                                                   1             74 ± 5                                                                              2.7                                          GVKAS.sup.1 #9                                                                          0            2330 ± 197                                                                           100                                                    0.1          2474 ± 163                                                                           106.2                                                  0.3           492 ± 41                                                                            21.1                                                   1             100 ± 7                                                                             4.3                                          GVKAS.sup.1 #13                                                                         0            1659 ± 107                                                                           100                                                    0.1          1242 ± 83                                                                            74.9                                                   0.3           220 ± 17                                                                            13.2                                                   1            NG        NG                                           GVKAR.sup.2 #7                                                                          0            1782 ± 125                                                                           100                                                    10           1121 ± 60                                                                            62.9                                                   30           1053 ± 65                                                                            59.1                                                   100           278 ± 19                                                                            15.6                                                   300           65 ± 3                                                                              3.6                                          GVKAR.sup.2 #9B                                                                         0            1800 ± 88                                                                            100                                                    10           1228 ± 67                                                                            68.2                                                   30            992 ± 79                                                                            55.1                                                   100           278 ± 13                                                                            15.4                                                   300           85 ± 5                                                                              4.7                                          GVKAR.sup.2 #25                                                                         0            1655 ± 60                                                                            100                                                    10            339 ± 25                                                                            20.5                                                   30            247 ± 16                                                                            14.9                                                   100           116 ± 7                                                                             7.0                                                    300          NG        NG                                           ______________________________________                                         .sup.1 Transformed cell line derived from transformed plant generated by      infection of tobacco leaf disks with Agrobacterium strain GV3850              containing a Ti plasmid carrying the sulfonylureasensitive Arabidopsis AL     gene.                                                                         .sup.2 Transformed cell line derived from transformed plant generated by      infection of tobacco leaf disks with Agrobacterium strain GV3850              containing a Ti plasmid carrying the sulfonylurearesistant Arabidopsis AL     gene.                                                                    

                  TABLE 18                                                        ______________________________________                                        ALS Enzyme Activities In Tobacco                                              Transformants which Received Arapidopois ALS Genes                                    (Δ0D530/mg/minute) × 100                                                       100 ppb     % Uninhibited                                Plant     No Herbicide                                                                             Clhorsulfuron                                                                             Activity                                     ______________________________________                                        GVKAS.sup.1 #13                                                                         1.172      0.074        6.3                                         GVKAS.sup.1 #15                                                                         1.244      0.048        3.9                                         GVKAS.sup.1 #18                                                                         0.553      0.014        2.5                                         GVKAR.sup.2 #7                                                                          1.724      0.766       44.4                                         GVKAR.sup.2 #9                                                                          0.694      0.412       59.4                                         GVKAR.sup.2 #9A                                                                         0.679      0.434       63.9                                         GVKAR.sup.2 #9B                                                                         1.081      0.714       66.0                                         GVKAR.sup.2 #25                                                                         0.861      0.341       39.6                                         ______________________________________                                         .sup.1 Transformed plant generated by infection of tobacco leaf disks wit     Agrobacterium strain GV3850 containing a Ti plasmid carrying the              sulfonylureasensitive Arabidopsis ALS gene.                                   .sup.2 Transformed plant generated by infection of tobacco leaf disks wit     Agrobacterium strain GV3850 containing a Ti plasmid carrying the              sulfonylurearesistant Arabidopsis ALS gene.                              

                  TABLE 19                                                        ______________________________________                                        Germination of Seeds Derived From Self-Pollinations                           of Tobacco Transformant Which Received Arabidopsis                            ALS Genes                                                                     Selfed Plant       Resistant                                                                              Sensitive                                         ______________________________________                                        GVKAS.sup.1 #4                                                                30 ppb Chlorsulfuron                                                                              0       45                                                300 ppb Chlorsulfuron                                                                             0       22                                                GVKAS.sup.1 #9                                                                30 ppb Chlorsulfuron                                                                              0       28                                                300 ppb Chlorsulfuron                                                                             0       28                                                GVKAS.sup.1 #13                                                               30 ppb Chlorsulfuron                                                                              0       62                                                300 ppb Chlorsulfuron                                                                             0       48                                                GVKAR.sup.2 #7                                                                300 ppb Chlorsulfuron                                                                            43        9                                                1000 ppb Chlorsulfuron                                                                           37       14                                                3000 ppb Chlorsulfuron                                                                           30       15                                                GVKAR.sup.2 #9                                                                300 ppb Chlorsulfuron                                                                            44       14                                                1000 ppb Chlorsulfuron                                                                           35       15                                                3000 ppb Chlorsulfuron                                                                           38       15                                                GVKAR.sup.2 #25                                                               300 ppb Chlorsulfuron                                                                            52       15                                                1000 ppb Chlorsulfuron                                                                           47       22                                                3000 ppb Chlorsulfuron                                                                           39       32                                                MMO Medium                                                                    Murashige and Skoog                                                           Major Salts                                                                   Murashige and Skoog                                                           Minor Salts                                                                   30 grams/L Sucrose                                                            100 mg/L i-Inositol                                                           0.4 mg/L Thiamine-HCL                                                         pH 5.8                                                                        0.8% Agar                                                                     ______________________________________                                         .sup.1 Transformed plant generated by infection of tobacco leaf disks wit     Agrobacterium GV3850 containing a Ti plasmid carrying the                     sulfonylureasensitive Arabidopsis ALS gene.                                   .sup.2 Transformed plant generated by infection of tobacco leaf disks wit     Agrobacterium strain GV3850 containing Ti plasmid carrying the                sulfonylurearesistant Arabidopsis ALS gene.                              

What is claimed is:
 1. A method for controlling the growth of undesiredvegetation growing at a locus where a plant has been cultivated, saidplant having been transformed with an isolated nucleic acid fragmentcomprising a nucleotide sequence encoding a plant acetolactate synthaseprotein which is resistant to a compound selected from the groupconsisting of sulfonylurea, triazolopyrimidine sulfonamide, andimidazolinone herbicides, said nucleotide sequence comprises at leastone sub-sequence which encodes one of the substantially conserved aminoacid sub-sequences designated A, B, C, D, E, F, and G, in FIG. 6, thenucleic acid fragment is further characterized in that at least one ofthe following conditions is met,(a) the nucleic acid fragment has asequence which encodes an amino acid sub-sequence A wherein ε₁ is anamino acid other than alanine, or ε₂ is an amino acid other thanglycine, (b) the nucleic acid fragment has a sequence which encodes anamino acid sub-sequence B wherein α₁ is an amino acid other thanproline, (c) the nucleic acid fragment has a sequence which encodes anamino acid sub-sequence C wherein δ₂ is an amino acid other thanalanine, (d) the nucleic acid fragment has a sequence which encodes anamino acid sub-sequence D wherein λ₁ is an amino acid other than lysine,(e) the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence E wherein γ₁ is an amino acid other than aspartic acid, (f)the nucleic acid fragment has a sequence which encodes an amino acidsub-sequence F wherein β₃ is an amino acid other than tryptophan, or β₈is an amino acid other than valine or β₇ is an amino acid other thanphenylalanine, and (g) the nucleic acid has a sequence which encodes anamino acid sub-sequence G wherein σ₁ is an amino acid other thanmethionine,said method comprising applying to the locus an effectiveamount of said herbicide.
 2. A method for controlling the growth ofundesired vegetation growing at a locus where a plant has beencultivated, said plant having been transformed with an isolated nucleicacid fragment comprising a nucleotide sequence encoding a plantacetolactate synthase protein, said nucleic acid fragment is capable ofbeing incorporated into a nucleic acid construct used to transform aplant containing wild-type acetolactate synthase protein which issensitive to a compound selected from the group consisting ofsulfonylurea, triazolopyrimidine sulfonamide, and imidazolinoneherbicides, said nucleic acid fragment having at least one pointmutation relative to the wild-type nucleic acid fragment encoding plantacetolactate synthase protein such that upon transformation with saidnucleic acid construct said plant is rendered resistant to theapplication of said herbicide compound; said method comprising applyingto the locus an effective amount of said herbicides.
 3. A methodaccording to claim 1 wherein the herbicide is a compound selected fromthe group consisting of ##STR12## wherein R is H or CH₃ ;J is ##STR13##R₁ is Cl, Br, NO₂, C₁ -C₄ alkyl, C₂ -C₄ alkenyl, CF₃, C₁ -C₄ alkoxy, C₁-C₄ haloalkoxy, C₃ -C₄ alkenyloxy, C₂ -C₄ haloalkenyloxy, C₃ -C₄alkynyloxy, CO₂ R₉, CONR₁₀ R₁₁, S(O)_(m) R₁₂, OSO₂ R₁₂, phenyl SO₂N(OCH₃ )CH₃, SO₂ NR₁₀ R₁₁, ##STR14## R₂ is H, Cl, Br, F, CH₃, NO₂, SCH₃,OCF₂ H, OCH₂ CF₃ or OCH₃ ; R₃ is Cl, NO₂, CO₂ CH₃, CO₂ C₂ H₅, SO₂N(CH₃)₂, SO₂ CH₃ or SO₂ C₂ H₅ ; R₄ is C₁ -C₃ alkyl, Cl, Br, NO₂, CO₂ R₉,CON(CH₃)₂, SO₂ N(CH₃)₂, SO₂ N(OCH₃)CH₃ or S(O)_(m) R₁₂ ; R₅ is C₁ -C₃alkyl, C₄ -C₅ cycloalkylcarbonyl, F, Cl, Br, NO₂, CO₂ R₁₄, SO₂ N(CH₃)₂,SO₂ R₁₂ or phenyl; R₆ is H, C₁ -C₃ alkyl, or CH₂ CH═CH₂ ; R₇ is H, CH₃,OCH₃, Cl or Br; R₈ is H, F, Cl, Br, CH₃, OCH₃, CF₃, SCH₃ or OCF₂ H; R₉is C₁ -C₄ alkyl, C₃ -C₄ alkenyl or CH₂ CH₂ Cl; R₁₀ is H or C₁ -C₃ alkyl;R₁₁ is H or C₁ -C₂ alkyl; R₁₂ is C₁ -C₃ alkyl; R₁₃ is H or CH₃ ; R₁₄ isC₁ -C₃ alkyl or CH₂ CH═CH₂ ; m is 0, 1 or 2; n is 1 or 2; Q is CH₂,CHCH₃ or NR₁₅ ; R₁₅ is H or C₁ -C₄ alkyl; P is O or CH₂ ; R₁₆ is H orCH₃ ; R₁₇ is C(O)NR₁₈ R₁₉ ; R₁₈ is H or CH₃ ; R₁₉ is CH₃ ; R₂₀ is H, Cl,F, Br, CH₃, CF₃, OCH₃ or OCF₂ H; R₂₁ is H or CH₃ ; X is CH₃, OCH₃, OC₂H₅ or NHCH₃ ; Y is CH₃, C₂ H₅, OCH₃, OC₂ H₅, OCF₂ H, OCH₂ CF₃, Cl, CH₂OCH₃ or cyclopropyl; Z is CH or N;and their agriculturally suitablesalts; provided that a) when Y is Cl then Z is CH and X is OCH₃ ; b)when Y is OCF₂ H, then Z is CH; c) when J is J-1 and R₁ is OSO₂ R₁₂ orphenyl, then Y is other than OCF₂ H; d) when J is J-2, then Y is otherthan OCF₂ H or OCH₂ CF₃ ; and e) when J is J-3 and R₄ is S(O)_(m) R₁₂,then Y is other than OCH₂ CF₃.
 4. The method of claim 3 whereinJ is J-1;R₁ is Cl, CH₃, C₁ -C₄ alkoxy, C₁ -C₂ haloalkoxy, allyloxy, propargyloxy,CO₂ R₉, CONR₁₀ R₁₁, SO₂ N(OCH₃)CH₃, SO₂ NR₁₀ R₁₁, S(O)_(m) R₁₂, OSO₂R₁₂, phenyl or ##STR15##
 5. The method of claim 3 whereinJ is J-2; R isH; and R₃ is SO₂ N(CH₃)₂, CO₂ CH₃ or CO₂ C₂ H₅.
 6. The method of claim 3whereinJ is J-3; R is H; and R₄ is CO₂ CH₃ or CO₂ C₂ H₅.
 7. The methodof claim 3 whereinJ is J-4; R is H; R₅ is Cl, Br, CO₂ CH₃, CO₂ C₂ H₅ or##STR16## R₆ is CH₃ ; and R₇ is H, Cl or OCH₃.
 8. The method of claim 3whereinJ is J-5; R is H; R₅ is CO₂ CH₃ or CO₂ C₂ H₅ ; and R₇ is H orCH₃.
 9. The method of claim 3 whereinJ is J-6; Q is CHCH₃ or NR₁₅ ; R isH; and R₈ is H, F, Cl, CH₃, OCH₃, CF₃ or SCH₃.
 10. The method of claim 3whereinJ is J-7; R is H; P is O; and R₈ is H, F, Cl, CH₃, OCH₃, CF₃ orSCH₃.
 11. The method of claim 3 whereinJ is J-8; R is H: R₁₆ is CH₃ ;and R₈ is H, F, Cl, CH₃, OCH₃, CF₃ or SCH₃.
 12. The method of claim 3whereinJ is J-9; R is H; and R₁₇ is C(O)N(CH₃)₂.
 13. The method of claim3 whereinR is H; R₁ is Cl, C₁ -C₄ alkoxy, OCF₂ H, OCH₂ CH₂ Cl, CO₂ R₉,CON(CH₃)₂, SO₂ N(CH₃)₂, SO₂ R₁₂ or OSO₂ R₁₂ ; and R₂ is H, Cl, CH₃, orOCH₃.
 14. A method according to claim 1 wherein the herbicide is acompound selected from the group consisting of ##STR17## wherein Ar is##STR18## R_(a) is C₁ -C₄ alkyl, F, Cl, Br, I, NO₂, S(O)_(p) R_(d),COOR_(e) or CF₃ ;R_(b) is H, F, Cl, Br, I, C₁ -C₄ alkyl or COOR_(e) ;R_(c) is H, C₁ -C₄ alkyl, F, Cl, Br, I, CH₂ OR_(d), phenyl, NO₂ orCOOR_(e) ; R_(d) is C₁ -C₄ alkyl; R_(e) is C₁ -C₄ alkyl, C₁ -C₄ alkenyl,C₁ -C₄ alkynyl, or 2-ethoxyethyl; V is H, C₁ -C₃ alkyl, allyl,propargyl, benzyl or C₁ -C₃ alkylcarbonyl; X₁, Y₁, and Z₁, areindependently H, F, Cl, Br, I, C₁ -C₄ alkyl, C₁ -C₂ alkylthio or C₁ -C₄alkoxy; and p is 0, 1 or
 2. 15. The method of claim 14 wherein V is H.16. The method of claim 15 whereinX₁ is H or CH₃ ; Y₁ is H; Z₁ is CH₃ ;and R_(a) and R_(c) are not simultaneously H.
 17. A method according toclaim 1 wherein the herbicide is a compound selected from the groupconsisting of ##STR19## wherein A is ##STR20## R_(f) is C₁ -C₄ alkyl;R_(g) is C₁ -C₄ alkyl or C₃ -C₆ cycloalkyl;A₁ is COOR_(i), CH₂ OH orCHO; R_(i) is H; C₁ -C₁₂ alkyl optionally substituted by C₁ -C₃ alkyl,C₃ -C₆ cycloalkyl or phenyl; C₃ -C₅ alkenyl optionally substituted byphenyl or 1-2 C₁ -C₃ alkyl, F, Cl, Br or I; or C₃ -C₅ alkynyl optionallysubstituted by phenyl or 1-2 C₁ -C₃ alkyl, F, Cl, Br or I; B is H;C(O)C₁ -C₆ alkyl or C(O)phenyl optionally substituted by Cl, NO₂ or OCH₃; X₂ is H, F, Cl, Br, I, OH or CH₃ ; Y₂ and Z₂ are independently H, C₁-C₆ alkyl, C₁ -C₆ alkoxy, F, Cl, Br, I, phenyl, NO₂, CN, CF₃ or SO₂ CH₃; X₃ is H, C₁ -C₃ alkyl, F, Cl, Br, I or NO₂ ; and L, M, Q and R_(h) areindependently H, F, Cl, Br, I, CH₃, OCH₃, NO₂, CF₃, CN, N(CH₃)₂, NH₂,SCH₃ or SO₂ CH₃ provided that only one of M or Q may be a substituentother than H, F, Cl, Br, I, CH₃ or OCH₃.
 18. The method of claim 17whereinB is H; and A₁ is COOR_(i).
 19. The method of claim 18,whereinR_(f) is CH₃ ; R_(g) is CH(CH₃)₂ ; X₂ is H; Y₂ is H or C₁ -C₃alkyl or OCH₃ ; Z₂ is H; X₃ is H, CH₃, Cl or NO₂ ; and L, M, Q and R_(h)are H.